Methods and Devices for Real-Time Diagnostic Testing (RDT) for Ebola and other Infectious Diseases

ABSTRACT

Methods, devices, systems, and kits for use in detecting and measuring infectious diseases are provided. In particular embodiments, methods, devices, systems, and kits for detecting and measuring Ebola virus, including Ebola Zaire strain virus, are provided. Devices and systems may be used within regions suffering from Ebola or other infections, providing local, rapid, and effective diagnosis of infectious diseases such as Ebola, improving treatment and reducing or preventing spread of such infectious diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of, and priority to, U.S. PatentApplication 62/061,671, filed Oct. 8, 2014; U.S. Patent Application62/062,808, filed Oct. 10, 2014; U.S. Patent Application 62/077,011,filed Nov. 7, 2014; U.S. Patent Application 62/077,016, filed Nov. 7,2014; U.S. Patent Application 62/094,848, filed Dec. 19, 2014; and U.S.Patent Application 62/094,856, filed Dec. 19, 2014, the entire contentsof which are hereby incorporated by reference in their entireties forall purposes.

BACKGROUND

Ebola is a highly contagious disease that causes the death of themajority of patients suffering from the disease. (The term “Ebola” isused to refer generally to all species and strains of hemorrhagicfever-inducing Ebola virus, including Zaire, Sudan, Tai Forest,Bundibugyo, and Reston.) The initial symptoms are similar to those ofmany viral disorders, most of which other viral disorders do not requirequarantine. An outbreak of Ebola in West Africa in 2014 highlighted theimportance of early diagnosis of Ebola victims in order to begintreatment of those victims, and in order to quickly isolate them so asto prevent the further spread of the disease. However, present methodsof detection and diagnosis of Ebola are time-consuming, expensive,complicated, and typically require laboratory equipment not oftenavailable in rural areas.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 7, 2015, isnamed 3042.601_SL.txt and is 51,058 bytes in size.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

SUMMARY

Applicant provides assays, devices, kits, methods, and systems foridentifying Ebola virus and for identifying blood markers that areindicative or characteristic of Ebola infection. These assays and assaymethods provide rapid results from small volume samples, such as smallvolume blood samples. These assays, devices, kits, methods, and systemsare effective when applied to small volume blood samples, such asfingerstick blood samples, including but not limited to samples as smallas 250 μL or less. These assays and assay methods are rapid, and provideresults within a short time, e.g., in three hours of less, within twohours or less, or within one hour or less from the time sample analysisbegins. In embodiments, these assays and assay methods provide resultswithin a short time, e.g., in three hours of less, or within two hoursor less, from the time a sample was obtained from a subject. In someembodiments, these assays, devices, kits, methods, and systems includenucleic acid assays for detection of Ebola Zaire virus (detected in theWest Africa outbreak in 2014) in EDTA-anti-coagulated whole blood. Theseassays, devices, kits, methods, and systems may be used and performed atpoint of service locations, including in rural locations or locationsremote from a hospital, clinic, or laboratory, as well as within aclinical laboratory or hospital.

Applicant provides assays, devices, kits, methods, and systems which maybe performed entirely within a region of active infection (a “hotzone”), such as a region where subjects suffering from Ebola infectionsare present. Samples obtained from subjects suspected of suffering anEbola infection may be obtained and analyzed within the hot zone usingautomatic sample analysis devices and systems (e.g., a sample processingunit (“SPU”)), so that no samples need leave the hot zone, therebyreducing possible further spread of the disease. Advantageous automaticsample processing devices or systems (for example, an SPU) containsamples entirely within the devices or systems during analysis followingloading the sample on or in the devices or systems; containing sampleswithin the system reduces or prevents spread of contamination and mayreduce or prevent the spread of disease that might otherwise occur dueto actions or events incident to sample analysis. An automatic sampleprocessing device or system (e.g., an SPU) may be enclosed in acontainer, or wrapped in a sheath, in order to reduce or preventcontamination of that device or system. Such a container or sheath mayprevent spread of infectious material or agents from one location toanother if the device or system is moved from one location to anotherlocation.

Applicant provides assays, devices, kits, methods, and systems whichutilize nucleic acid assays along with assays for antibodies to theEbola virus. In embodiments, Applicant provides assays, devices, kits,methods, and systems which utilize i) nucleic acid assays, ii) assaysfor antibodies to the Ebola virus, and iii) electrolyte assays. Inembodiments, nucleic acid assays may comprise isothermal nucleic acidassays. In embodiments, isothermal nucleic acid assays includeisothermal nucleic acid assays as described in U.S. patent applicationSer. No. 14/214,850, filed Mar. 15, 2014; in international patentapplication PCT/US2014/030034, filed Mar. 15, 2014; and in internationalpatent application PCT/US2014/056151, filed Sep. 17, 2014; these methodsare collectively termed “TNAA” methods. In embodiments, assays forantibodies to the Ebola virus comprise IgM and IgG assays for antibodiesto the Ebola virus. In embodiments, electrolyte assays comprise sodiumassays, or potassium assays, or sodium and potassium assays. Thus, inembodiments, Applicant provides assays, devices, kits, methods, andsystems which utilize i) nucleic acid assays, ii) IgM and IgG assays forantibodies to the Ebola virus, and iii) Sodium and Potassium assays. Inembodiments, Applicant provides assays, devices, kits, methods, andsystems which utilize i) TNAA nucleic acid assays, ii) IgM and IgGassays for antibodies to the Ebola virus, and iii) Sodium and Potassiumassays. Such assays are typically performed on blood samples (e.g.,fingerstick blood samples) obtained from subjects suspected of sufferingfrom Ebola; however, in embodiments, such assays may be performed onurine or other bodily fluid samples obtained from a subject.

These assays, devices, kits, methods, and systems are suitable forevaluating patients with signs and symptoms of Ebola infection and forelectrolyte balance management; evaluation of electrolytes allowscaregivers to quickly assess patients for dehydration, and is believedto help protect caregivers from unnecessary Ebola exposure risk byexpediting the assessment of whether oral or intravenous hydration isappropriate (e.g., avoidance of intravenous hydration where possible isbelieved to reduce Ebola exposure risk to caregivers).

The assays, devices, kits, methods, and systems disclosed herein may beused with blood samples collected by capillary means, e.g., with samplesobtained using single-use fully retractable lancets. Thus, unlike priormethods which require personnel trained in performing phlebotomy, theassays, devices, kits, methods, and systems do not require needles anddo not require trained personnel. Furthermore, the systems, devices, andkits can be deployed and used in any area, including rural areas, whereEbola is present or spreading, effective to provide local and real-timeassessment, treatment and quarantine in a “hot zone” where the need isgreatest. In embodiments, the assays, devices, kits, methods, andsystems disclosed herein facilitate real-time reporting and modeling ofcases through real-time connectivity to self-learning epidemiologicalmodels and software systems; e.g., assays may be run in configurationsthat enable remote connectivity through electronic links (e.g.,satellite link, cellular network, landline, or other communicationlink).

In embodiments, the assays, devices, kits, methods, and systems may beused in conjunction with further assays useful for triage of patients toidentify those suffering from Ebola from those suffering from otherdiseases. Such other assays useful for such triage include assays fordetecting or measuring other pathogens causing other fevers of unknownorigin, molecular tests for multiple strains of Ebola virus, assays forhemoglobin, and iron tests for anemia. In embodiments, assays for feversof unknown origin may include but are not limited to assays for Lassafever, malaria, cholera, typhoid fever, Marburg, dengue I, II, III andIV, chikungunya, West Nile, and meningitis. Triage of patients isbelieved to enable the rapid separation of Ebola patients from otherpatients, and to reduce the risk of Ebola infection to such otherpatients. Anemia assays may be useful to aid in triaging of patients(e.g., to determine whether or not a subject suffers from hookworm orother conditions, and so are at risk of dying of anemia for that reasonas well, e.g., before being able to get treated for Ebola infectionitself).

Accordingly, in embodiments, Applicants disclose an assay for thedetection of Ebola Zaire virus (detected in the West Africa outbreak in2014) comprising a TNAA assay that is for the detection of Ebola Zairevirus in whole blood or plasma specimens (e.g., in EDTA-anti-coagulatedspecimens). In embodiments, such assays include assays for detectinganti-Ebola antibodies in heparin-anticoagulated plasma specimens or inEDTA-anti-coagulated whole blood specimens. In embodiments, such assaysinclude assays for detecting and measuring electrolyte levels, such assodium and potassium levels in heparin-anticoagulated plasma specimens.Thus, Applicant discloses simultaneous or contemporaneous performance ofi) a TNAA assay for Ebola Zaire virus, ii) IgM and IgG assays for thequalitative detection of IgM and IgG antibodies for Ebola virus, andiii) the quantitative measurement of levels of sodium and potassium fromwhole blood or plasma specimens (e.g., EDTA-anti-coagulated, orheparin-anti-coagulated whole blood or plasma, which may be extractedfrom whole blood specimens). Optionally, some embodiments may includeAntigen ELISA assay for detection of the virus. In one embodiment, theTNAA assay may be used to detect for the virus in a window of 3 to 10days after the onset of fever in the subject. In embodiments, one ormore of such assays are performed within an automatic sample analysisdevice or system (termed herein “SPU”). In embodiments,heparin-anti-coagulated plasma is extracted from whole blood in an SPU.IN embodiments, samples provided herein may contain an anticoagulant,such as heparin or EDTA.

Applicant discloses devices, including automatic sample analysis devices(e.g., an SPU) for performing the assays disclosed herein.

Applicant discloses systems, the systems including cartridges andincluding automatic sample analysis devices (e.g., an SPU) forperforming the assays disclosed herein, the cartridges being configuredfor use with the automatic sample analysis devices. A system asdisclosed herein may include a communication device, or communicationlink, or other electronic connection configured to provide information(e.g., protocols) to the automatic sample analysis device, or configuredto transmit data or other information from the automatic sample analysisdevice, or both. A cartridge may include reagents for use in processingor testing a sample, disposables for use in processing or testing asample, or other materials. For example, a cartridge may includereagents for use in processing or testing a sample for the presence ofviral nucleic acids (e.g., Ebola RNA); may include reagents for use inprocessing or testing a sample for the presence of viral amino acids(e.g., Ebola nucleoprotein, or other proteins and proteinaceous materialindicative of Ebola infection); may include reagents for use inprocessing or testing a sample for the presence of antibodies to a virus(e.g., antibodies to Ebola virus antigens); may include reagents for usein processing or testing a sample for the presence of electrolytes(e.g., for detecting sodium or potassium level abnormalities in asubject's blood); and combinations thereof. A cartridge may includevessels, such as, e.g., reagent vessels, mixing vessels, waste vessels,or other vessels, for use in processing or testing a sample. A cartridgemay include disposables for use in processing or testing a sample. Acartridge may include cuvettes, and may include other components,elements, and materials for use in processing or testing a sample. Acartridge may include at least one sample. Optionally, the cartridgeincludes at least one or more slots for the insertion of one or morecontainers that may contain one or more samples.

Applicant discloses kits, the kits including reagents for performing theassays disclosed herein. In embodiments, the kits may include cartridgesconfigured for use with the automatic sample analysis devices forperforming the assays disclosed herein. In embodiments, the kits mayinclude sample collection devices for performing the assays disclosedherein. In embodiments, the kits containing reagents may includecartridges, and, in embodiments, the reagents may be carried on thecartridges. In embodiments, the reagents carried on cartridges mayinclude reagents for assays for detecting nucleic acids (e.g., Ebolavirus nucleic acids); for detecting proteins or proteinaceaous material(e.g., reagents for detecting antibodies to Ebola virus, for detectingEbola virus antigens, or both); for detecting electrolytes (e.g., fordetecting sodium, potassium, or other electrolytes); and combinationsthereof. In embodiments, the kits may include sample containersconfigured for use with the cartridges, or with the automatic sampleanalysis devices, or both, for performing the assays disclosed herein.

Such assays may be applied to any subject or any blood sample; forexample, such assays may be usefully applied to samples obtained fromindividuals in affected areas with signs and symptoms of Ebola virusinfection or who are at risk for exposure or may have been exposed tothe Ebola Zaire virus (detected in the West Africa outbreak in 2014) inconjunction with epidemiological risk factors. In embodiments, theassays, devices, kits, methods, and systems disclosed herein may be usedin areas where subjects may be at risk of contracting Ebola, e.g., inareas where patients may present for diagnosis and treatment of Ebolainfection.

The assays, devices, systems, methods, and kits disclosed herein providemany advantages and benefits. A chief benefit associated with thepresent Ebola Zaire TNAA assays, along with the Ebola antibody andelectrolyte assays disclosed herein, is the ability to perform continuedtesting of human specimens for Ebola virus, even in rural and remotelocations, and even outside of clinical, hospital, or laboratoryenvironments. The testing of human specimens for Ebola virus asdisclosed herein provide diagnostic, clinical, and public healthbenefits, in addition to providing better tools for further research.True positive results provide confirmatory support for the diagnosis ofan Ebola virus infection. True positive results also support thedecision to isolate patients in negative-pressure rooms, if available,and employ practical viral hemorrhagic fever isolation precautions.Also, establishing the Ebola virus as the true cause of the patient'ssymptoms prevents further workup for other possible causes and allowssaving of other healthcare resources. True negative results benefit bothphysicians and patients by ruling out a diagnosis and allowing otherpossible illnesses to be pursued.

The assays additionally benefit public health. If people infected withthe Ebola virus are immediately isolated from other people andappropriate precautions are taken, this can prevent others from gettingsick. By having this test done, the chance of spreading the virus toothers is reduced. Also, using the test can help healthcare providerslearn more about the Ebola virus and stop its spread.

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key features oressential features of the claimed subject matter, nor is it intended tobe used to limit the scope of the claimed subject matter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic outline of systems for detecting Ebola virusand related markers of Ebola as described herein. FIG. 1 depicts thepre-analytical (sample processing, performed by the sample processingdevices and systems described herein (termed “SPU”, and as controlled bylaboratory automation systems, termed herein “LAS”), analytical (reportgeneration, performed by the LAS), and post-analytical (reporttransmission, performed by the LAS) parts of the devices and systemsdisclosed herein, and of the pre-analytical, analytical, andpost-analytical steps of the methods disclosed herein.

FIG. 2A provides a schematic description of TNAA nucleic acidpurification steps. In embodiments, such steps are performed in anautomatic sample analysis device or system (e.g., an SPU) and may beperformed in the field (e.g., in rural or other locations, and notnecessarily within a laboratory, clinic, or hospital setting.

FIG. 2B shows a simplified depiction of one embodiment of SPU and oneembodiment of a cartridge as described herein.

FIG. 3 illustrates experiments performed which show limits of detection(LOD) of the TNAA nucleic acid assay as applied to Ebola Zaire viralnucleoprotein RNA. As shown in the figure, titration of synthetic RNAtarget encoding Ebola Zaire nucleoprotein shows an LOD as low as 10copies/uL. The y-axis shows the time of detection based on thefluorescent signal recorded in real-time during the TNAA amplificationreaction (i.e., the amplification reaction performed in a TNAA nucleicacid assay). The cutoff for assessing a positive reaction was 45.6minutes.

FIG. 4 illustrates experiments performed which show amounts ofcross-reactivity of the TNAA nucleic acid assay as applied to EbolaZaire viral nucleoprotein and potential cross-reactants. The crossreactivity of the TH-EZN reaction to other pathogens is shown here. Nofalse positive reaction was seen. The positive control is labeled “EbolaZaire 100/cp/uL”. The non-template control is labeled “NTC”. The y-axisshows the time of detection based on the fluorescent signal recorded inreal-time during the TNAA amplification reaction. The cutoff forassessing a positive reaction was 45.6 minutes.

FIG. 5. This figure illustrates the specificity of the TNAA nucleic acidassays. The specificity of the TH-EZN reaction was tested by adding thelisted pathogens to the sample in addition to the positive controlsynthetic templates. No false negative reactions were seen. Ebola Zairevirus RNA was used as a positive control. The non-template control islabeled “NTC”. The y-axis shows the time of detection based on thefluorescent signal recorded in real-time during the TNAA amplificationreaction. The cutoff for assessing a positive reaction was 45.6 minutes.

FIG. 6. The effect of interfering substances was tested for the TH-EZNreaction in two ways. First interfering substances were added to thesample in addition to the positive control synthetic templates to see ifthe substances interfered with the reaction (“Inhibits True Positive”).Second, interfering substances were added to a sample without anytemplate (NTC) to assess if a non-specific reaction occurred (“CausesFalse Positive”). Each substance was tested at two levels.

FIG. 7A shows results of assays using anti-Ebola IgG antibodies thatwere tested on spiked plasma samples. The data shown in FIG. 7Ademonstrates the performance of the Ebola IgG Assays. FIG. 7A providesresults comparing in-house (negative) EDTA-anticoagulated plasma sampleswith spiked samples. All samples were spiked with human anti-ZEBOV IgGto a nominal concentration of 1 μg/ml,

FIG. 7B compares assay results from negative and spiked samples; thefigure shows signals from assays run with in-house (negative)EDTA-anticoagulated plasma samples (squares) and spiked samples. Allsamples were spiked with human anti-ZEBOV IgG.

FIG. 7C provides a Table that summarizes cross reactivity data fromEbola IgG Assays.

FIG. 8A shows results of assays using anti-Ebola antibodies were testedon spiked plasma samples. FIG. 8A provides results comparing in-house(negative) EDTA-anticoagulated plasma samples with spiked weak positivesamples. All samples were spiked with human anti-ZEBOV IgM to a nominalconcentration of 0.3 μg/ml.

FIG. 8B compares assay results from negative and spiked samples; thefigure shows signals from assays run with in-house (negative)EDTA-anticoagulated plasma samples (squares) and spiked samples. Allsamples were spiked with human anti-ZEBOV IgM.

FIG. 9A shows dose-response results from assays with recombinant ZEBOVGP antigen spiked pooled normal serum calibrators.

FIG. 9B provides a Table that summarizes cross reactivity data fromZEBOV GP antigen ELISA Assays.

DETAILED DESCRIPTION

Description and disclosure of examples of reagents, assays, methods,kits, devices, and systems which may use, or be used with, assays,devices, systems, kits, and methods disclosed herein may be found, forexample, in U.S. Pat. No. 8,088,593; U.S. Pat. No. 8,380,541; U.S. Pat.No. 8,435,738; U.S. Pat. No. 8,475,739; U.S. Pat. No. 8,840,838; U.S.patent application Ser. No. 13/769,820, filed Feb. 18, 2013; U.S. patentapplication Ser. No. 14/183,503, filed Feb. 18, 2014; U.S. patentapplication Ser. No. 14/214,850, filed Mar. 15, 2014; InternationalPatent Application PCT/US2014/030034, filed Mar. 15, 2014; InternationalPatent Application PCT/US2014/056151, filed Sep. 17, 2014; U.S. patentapplication Ser. No. 13/769,798, filed Feb. 18, 2013; U.S. patentapplication Ser. No. 13/769,779, filed Feb. 18, 2013; U.S. patentapplication Ser. No. 13/244,947 filed Sep. 26, 2011; PCT/US2012/57155,filed Sep. 25, 2012; U.S. patent application Ser. No. 13/244,946, filedSep. 26, 2011; U.S. patent application Ser. No. 13/244,949, filed Sep.26, 2011; and U.S. application Ser. No. 13/945,202, filed Jul. 18, 2013,the disclosures of which patents and patent applications are all herebyincorporated by reference in their entireties.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. It may be notedthat, as used in the specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “a material”may include mixtures of materials, reference to “a compound” may includemultiple compounds, and the like. References cited herein are herebyincorporated by reference in their entirety, except to the extent thatthey conflict with teachings explicitly set forth in this specification.

Acronyms used herein typically have their customary meanings. Someacronyms as used herein include: ribonucleic acid (RNA);dexoyribonucleic acid (DNA); limits of detection (LOD); SampleProcessing Unit (SPU); Laboratory Automation System (LAS); ClinicalLaboratory Improvements Amendments (CLIA); polymerase chain reaction(PCR); reverse transcriptase PCR (RT-PCR); quantitative RT-PCR(Q-RT-PCR); personal protective equipment (PPE); real-time diagnostic(RDT); alkaline phosphatase (ALP); ethylene diamine tetra acetic acid(EDTA); Ebola Virus, Zaire strain (ZEBOV); Tetraphenylborate (TPB);Sodium Tetraphenylborate (NaTPB); Nucleic acid assay (NAA); isothermalnucleic acid assay (TNAA, or TNAA assay, or TNAA nucleic acid assay);Ebola virus Glycoprotein (GP); Ebola Zaire virus nucleoprotein (TH-EZN);Ebola Zaire virus glycoprotein (TH-EZG); RNA Spike-in Control(TH-RNA-SIC) assay; and Human Centromeric Repeat (TH-HCR) assay.

In this specification and in the claims which follow, reference may bemade to a number of terms which shall be defined to have the followingmeanings:

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.For example, if a device optionally contains a feature for a samplecollection unit, this means that the sample collection unit may or maynot be present, and, thus, the description includes both structureswherein a device possesses the sample collection unit and structureswherein sample collection unit is not present.

As used herein, the terms “substantial” means more than a minimal orinsignificant amount; and “substantially” means more than a minimally orinsignificantly. Thus, for example, the phrase “substantiallydifferent”, as used herein, denotes a sufficiently high degree ofdifference between two numeric values such that one of skill in the artwould consider the difference between the two values to be ofstatistical significance within the context of the characteristicmeasured by said values. Thus, the difference between two values thatare substantially different from each other is typically greater thanabout 10%, and may be greater than about 20%, preferably greater thanabout 30%, preferably greater than about 40%, preferably greater thanabout 50% as a function of the reference value or comparator value.

The term “nucleic acid” refers to nucleotides and nucleosides which makeup, for example, deoxyribonucleic acid (DNA) macromolecules andribonucleic acid (RNA) macromolecules. Nucleic acids may be identifiedby the base attached to the sugar (e.g., deoxyribose or ribose); as usedherein, the following abbreviations for these bases are used torepresent nucleic acids in sequence listings identifying and describingtheir structures (either upper-case or lower-case may be used).

TABLE 1A Base (in Nucleic Acid) Letter Code Adenine A Thymine T GuanineG Cytosine C Uracil U

The terms “peptide”, “polypeptide”, “proteinaceous material”, and“protein” may be used interchangeably to refer to molecules comprised ofamino acids linked by peptide bonds. Individual amino acids may betermed “residues” of a polypeptide or protein. The amino acid sequencesof polypeptides disclosed herein may be identified by SEQ ID NO:presented as a string of letters, where the letters have the followingmeanings:

TABLE 1B Amino Acid 3-Letter Code 1-Letter Code Alanine Ala A ArginineArg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamic acidGlu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile ILeucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F ProlinePro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr YValine Val V

As used herein, a “sample” may be but is not limited to a blood sample,or a portion of a blood sample, may be of any suitable size or volume,and is preferably of small size or volume. A sample may be, for example,a blood sample (e.g., a sample obtained from a fingerstick, or fromvenipuncture, or an arterial blood sample), a urine sample, a biopsysample, a tissue slice, stool sample, or other clinical sample; a watersample, a soil sample, a food sample, an air sample; or other sample. Ablood sample may comprise, e.g., whole blood, plasma, or serum. In someembodiments of the assays and methods disclosed herein, measurements maybe made using a small volume blood sample, or no more than a smallvolume portion of a blood sample, where a small volume comprises no morethan about 5 mL; or comprises no more than about 3 mL; or comprises nomore than about 2 mL; or comprises no more than about 1 mL; or comprisesno more than about 500 μL; or comprises no more than about 250 μL; orcomprises no more than about 100 μL; or comprises no more than about 75μL; or comprises no more than about 50 μL; or comprises no more thanabout 35 μL; or comprises no more than about 25 μL; or comprises no morethan about 20 μL; or comprises no more than about 15 μL; or comprises nomore than about 10 μL; or comprises no more than about 8 μL; orcomprises no more than about 6 μL; or comprises no more than about 5 μL;or comprises no more than about 4 μL; or comprises no more than about 3μL; or comprises no more than about 2 μL; or comprises no more thanabout 1 μL; or comprises no more than about 0.8 μL; or comprises no morethan about 0.5 μL; or comprises no more than about 0.3 μL; or comprisesno more than about 0.2 μL; or comprises no more than about 0.1 μL; orcomprises no more than about 0.05 μL; or comprises no more than about0.01 μL.

As used herein, a “fingerstick” or a “fingerstick sample” is a bloodsample obtained by a small puncture in the skin, typically, although notnecessarily, on a finger (e.g., such a puncture may be made on any skinsurface, including a toe, a heel, an arm, a leg, or other body portion).A puncture for a fingerstick may be made, e.g., by a lancet, a needle, asharp probe, or any other suitable instrument or implement.

As used herein, the term “point of service location” may includelocations where a subject may receive a service (e.g. testing,monitoring, treatment, diagnosis, guidance, sample collection, IDverification, medical services, non-medical services, etc.), and mayinclude, without limitation, a subject's home, a subject's business, thelocation of a healthcare provider (e.g., doctor), hospitals, emergencyrooms, operating rooms, clinics, health care professionals' offices,laboratories, retailers [e.g. pharmacies (e.g., retail pharmacy,clinical pharmacy, hospital pharmacy), drugstores, supermarkets,grocers, etc.], transportation vehicles (e.g. car, boat, truck, bus,airplane, motorcycle, ambulance, mobile unit, fire engine/truck,emergency vehicle, law enforcement vehicle, police car, or other vehicleconfigured to transport a subject from one point to another, etc.),traveling medical care units, mobile units, schools, day-care centers,security screening locations, combat locations, health assisted livingresidences, government offices, office buildings, tents, bodily fluidsample acquisition sites (e.g. blood collection centers), sites at ornear an entrance to a location that a subject may wish to access, siteson or near a device that a subject may wish to access (e.g., thelocation of a computer if the subject wishes to access the computer), alocation where a sample processing device receives a sample, or anyother point of service location described elsewhere herein.

Nucleic Acid Amplification Methods

The TNAA nucleic acid amplification method includes an isothermal methodthat provides rapid qualitative detection and identification ofpathogens from clinical samples. In embodiments, a pre-amplificationstep may be included before the isothermal amplification; such apre-amplification step may increase the overall sensitivity of theoverall TNAA method.

For example, in order to further enhance the sensitivity of the TNAAassay, a pre-amplification step may be performed via RT-PCR on an SPU.PCR primers are designed to amplify the target gene for the subsequentisothermal amplification reactions used for specific detection. TheRT-PCR reaction is conducted by thermal cycling, and the final productof this reaction is used for the subsequent isothermal amplification andspecific detection conducted subsequently. Thus, in embodimentsutilizing thermal cycling nucleic acid pre-amplification and isothermalnucleic acid amplification, steps may include, for example,pre-amplification by RT-PCR of an extracted RNA sample, resulting ingreatly increasing copy numbers in the original extracted sample. Theresulting pre-amplified product forms the starting sample for the TNAAreaction. Subsequent processing of the pre-amplified product by TNAA(i.e., without further thermal cycling) provides further amplification,and provides detection, of the target molecules.

Methods for nucleic acid amplification which do not require thermalcycling are described in U.S. Patent Application 61/800,606, filed Mar.15, 2013, incorporated by reference herein in its entirety. Such methodsmay be used to detect nucleic acid markers of disease, such asrespiratory disease, in small-volume samples in short periods of time.Such methods are discussed below, and examples of results obtained withsuch methods, from small samples and in short periods of time, arepresented in the Figures and Examples disclosed herein. In thefollowing, such methods are termed “non-cycling amplification methods.”

Non-cycling amplification methods of nucleic acid amplification may beapplied to double-stranded DNA. However, target nucleic acid moleculesneed not be limited to double-stranded DNA targets; for example,double-stranded DNA for use in non-cycling amplification methodsdescribed herein may be prepared from viral RNA, or mRNA, or othersingle stranded RNA target sources, by reverse transcriptase. In furtherexample, double-stranded DNA for use in non-cycling amplificationmethods described herein may be prepared from single-stranded DNAtargets by DNA polymerase. Such methods may be applied as an initialstep, prior to application of the non-cycling amplification methodsdiscussed below.

Amplification of a double-stranded DNA target, for example, begins witha primary double-stranded DNA to be amplified (termed the “primarynucleic acid” in the following). The primary nucleic acid contains atarget region termed a template region; the template region has atemplate sequence. Such a double-stranded template region contains afirst DNA strand and a complementary second DNA strand, and includes a5′ terminal nucleotide in one strand and a 3′ terminal nucleotide in theother strand that are complementary to each other.

A first primer and a second primer are provided which each havetemplate-binding regions and tail regions; the primer template-bindingregions are complementary to the target template regions. The tailregions of the primers may contain three components: a) the 5′ terminalnucleotide of the primer, b) an innermost nucleotide, wherein theinnermost nucleotide is downstream from the 5′ terminal nucleotide, andc) a middle section between the 5′ terminal nucleotide and the innermostnucleotide, comprising one or more nucleotides. In addition, at leastportions of the two primer tail regions may be complementary to eachother when properly aligned.

It should be noted that although the tail region of the second primermay contain a nucleotide sequence which is complementary to thenucleotide sequence of the tail region of the first primer, typically,products formed by the annealing of the first primer and second primerare not desirable or useful for methods or compositions provided herein.Accordingly, in some embodiments, steps may be taken to minimize theformation of first primer—second primer annealed products. Such stepsmay include, for example, not pre-incubating a first primer and a secondprimer under conditions where the primers may anneal for an extendedperiod of time before initiating a method provided herein.

The primary nucleic acid may be treated with a polymerase and a firstcopy of the first primer under conditions such that the template-bindingregion of the first copy of the first primer anneals to the first strandof the nucleic acid template. Under these conditions, an extensionproduct of the first copy of the first primer is formed. The polymerase,which may have strand displacement activity, may catalyze the formationof the extension product of the first copy of the first primer. Thefirst copy of the first primer may be covalently linked to thesynthesized extension product, such that the first copy of the firstprimer (which is complementary to the first strand of the nucleic acidtemplate) becomes part of the molecule described herein as the“extension product of the first copy of the first primer.” Thetemplate-binding region but not the tail region of the first copy of thefirst primer anneals to the first strand of the nucleic acid template.Examples of conditions suitable for polymerase-based nucleic acidsynthesis are known in the art and are provided, for example, inMolecular Cloning: A Laboratory Manual, M. R. Green and J. Sambrook,Cold Spring Harbor Laboratory Press (2012), which is incorporated byreference herein in its entirety.

The extension product of the first copy of the first primer may betreated with a polymerase (which may have strand displacement activity)and with the second primer under conditions such that thetemplate-binding region of the second primer anneals to the extensionproduct of the first copy of the first primer. In this way, an extensionproduct of the second primer may be formed. The polymerase may displacethe first strand of the nucleic acid template from the extension productof the first copy of the first primer during the synthesis of theextension product of the second primer. The second primer may becovalently linked to the synthesized extension product, such that thesecond primer becomes part of the molecule described herein as the“extension product of the second primer.” The extension product of thesecond primer is complementary to the extension product of the firstcopy of the first primer. The template-binding region but not the tailregion of the second primer may anneal to the extension product of thefirst copy of the first primer when the second primer anneals to theextension product of the first copy of the first primer.

The extension product of the second primer may be treated with apolymerase (which may have strand displacement activity) and a secondcopy of the first primer so as to form an extension product of thesecond copy of the first primer. During the generation of the extensionproduct of the second copy of the first primer, the second copy of thefirst primer may be covalently linked to the synthesized extensionproduct, such that the second copy of the first primer becomes part ofthe molecule described herein as the “extension product of the secondcopy of the first primer.” The extension product of the second copy ofthe first primer is complementary to the extension product of the secondprimer.

Generation of the extension product of the second copy of the firstprimer may result in the generation of a molecule comprising theextension product of the second copy of the first primer and theextension product of the second primer, which may be referred to hereinas the “secondary nucleic acid.” A secondary nucleic acid may comprisethe 3′ terminal region of the extension product of the second primer(and the complement thereof) and may comprise the 3′ terminal region ofthe extension product of the second copy of the first primer (and thecomplement thereof). Secondary nucleic acid molecules include sequencesof the template region adjacent to tail sequences. In embodiments,double-stranded nucleic acids are produced in which complementarytemplate and tail region sequences line up. In practice, multiple copies(e.g., two or more) of the secondary nucleic acid are produced by anyprocess whereby a nucleic acid having the general structure of thesecondary nucleic acid may be generated, including by practice ofnon-cycling amplification methods discussed herein.

Thus, pairs of copies of the secondary nucleic acid may be provided.Further numbers of copies may then be generated, for example, byrepetition of the foregoing steps and methods. For example, the fullprocess as described above for generating a secondary nucleic acid froma primary nucleic acid may be repeated two times, in order to generate atwo pairs of copies of the secondary nucleic acid; further repetitionsmay be performed to amplify the number of copies further, e.g., toexponentially amplify the number of copies (e.g., by powers of two).

In addition, since the secondary nucleic acid molecules includesequences of the template region adjacent to tail sequences, partiallydouble-stranded nucleic acids may be produced in which tail regionsequences hybridize and line up. Since these tail region sequences areattached to single-stranded template regions, a cross-over structurehaving two nucleic acid strands together held by the hybridized tailregion sequences is produced. These cross-over structures may beextended by a polymerase to form extension products of both componentstrands. These extension products which may be referred to as“concatemer strands.” Two concatemer strands may be annealed together,and may be collectively referred to as a concatemer; such concatemersmay contain two or more copies of the nucleic acid template.

In some embodiments, even longer concatemers may be formed. For example,concatemers may anneal together; or two concatemer molecules may form across-over structure similar to those formed by the shorter moleculestermed concatemer strands, as discussed above, followed by a largerconcatemer molecule containing four copies of the nucleic acid template.In another example, a secondary nucleic acid and a concatemer may form across-over structure, followed by a larger concatemer moleculecontaining three copies of the nucleic acid template. In someembodiments, multiple different concatemers of multiple differentlengths may be simultaneously generated.

Thus, concatemers generated according to such methods may be of anylength of nucleotides. In some embodiments, concatemer moleculesgenerated herein may be at least 30, 40, 50, 60, 70, 80, 90, 100, 150,200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000,4000, 5000, 6000, 7000, 8000, 9000, 10,000, 15,000, 20,000, or 25,000nucleotides in length. Concatemers generated according to such methodsmay contain any number of copies of a nucleic acid template. In someembodiments, concatemer molecules generated herein may contain at least2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 copies of a nucleic acidtemplate. Further examples are provided, and greater detail of these andother examples, is provided in U.S. Patent Application 61/800,606, filedMar. 15, 2013.

Detection of Reactions

Progress of a method provided herein may be monitored in multipledifferent ways. In one embodiment, a reaction may be assayed for anucleic acid amplification product (e.g. for the level of the product orthe rate of its generation). In another embodiment, a reaction may beassayed for the activity of a polymerase along a nucleic acid template(e.g. for movement of a polymerase along a template strand). Thus, insome embodiments, events of a method provided herein may observed due tothe accumulation of product from a method (which may be during or aftercompletion of steps of the method), or due to detectable eventsoccurring during the steps of a method.

The presence of amplified nucleic acids can be assayed, for example, bydetection of reaction products (amplified nucleic acids or reactionby-products) or by detection of probes associated with the reactionprogress.

In some embodiments, reaction products may be identified by staining theproducts with a dye. In some embodiments, a dye may have greaterfluorescence when bound to a nucleic acid than when not bound to anucleic acid. In some embodiments, a dye may intercalate with adouble-stranded nucleic acid or it may bind to an external region of anucleic acid. Nucleic acid dyes that may be used with methods andcompositions provided herein include, for example, cyanine dyes,PicoGreen®, OliGreen®, RiboGreen®, SYBR® dyes, SYBR® Gold, SYBR® GreenI, SYBR® Green II, ethidium bromide, dihydroethidium, BlueView™, TOTO®dyes, TO-PRO® dyes, POPO® dyes, YOYO® dyes, BOBO® dyes, JOJO® dyes,LOLO® dyes, SYTOX® dyes, SYTO® dyes, propidium iodide, hexidium iodide,methylene blue, DAPI, acridine orange, quinacrine, acridine dimers,9-amino-6-chloro-2-methoxyacridine, bisbenzimide dyes, Hoechst dyes,7-aminoactinomycin D, actinomycin D, hydroxystilbamidine, pyronin Y,Diamond™ dye, GelRed™, GelGreen™ and LDS 751.

In some embodiments, reaction products may be identified by analysis ofturbidity of amplification reactions for example, where increasedturbidity is correlated with formation of reaction products and reactionby-products (e.g. pyrophosphate complexed with magnesium).

In some embodiments, reaction products may be identified by separating areaction performed according to a method herein by gel electrophoresis,followed by staining of the gel with a dye for nucleic acids. The dyemay be any nucleic acid dye disclosed herein or otherwise known in theart.

In some embodiments, any method or composition known in the art for thedetection of nucleic acids or processes associated with the generationof nucleic acids may be used with methods and compositions providedherein.

In some embodiments, a nucleic acid probe which contains a nucleotidesequence complementary to a portion of a nucleic acid template strand(or strand having a similar or identical sequence) and which containsone or both of a fluorescent reporter (fluorophore) and a quencher areincluded in a reaction provided herein.

In an example, a nucleic acid probe may contain a fluorescent reporterat its 5′ or 3′ terminus, and a quencher at the other terminus.

In another example, a nucleic acid probe may contain a fluorescentreporter at its 5′ or 3′ terminus, and it may be annealed to a nucleicacid primer containing a quencher. The nucleic acid primer containing aquencher may contain the quencher at a position in the primer such thatwhen the nucleic acid probe is annealed to the primer, the fluorescentreporter is quenched.

In probes containing a fluorescent reporter and quencher pair, thefluorescent reporter and quencher may be selected so that the quenchercan effectively quench the reporter. In some embodiments, a fluorescentreporter is paired with a quencher where the emission maximum of thefluorescent reporter is similar to the absorption maximum of thequencher. Fluorphores that may be used as the fluorescent reporterinclude, for example, CAL Fluor Gold, CAL Fluor Orange, Quasar 570, CALFluor Red 590, CAL Fluor Red 610, CAL Fluor Red 610, CAL Fluor Red 635,Quasar 670 (Biosearch Technologies), VIC, NED (Life Technologies), Cy3,Cy5, Cy5.5 (GE Healthcare Life Sciences), Oyster 556, Oyster 645(Integrated DNA Technologies), LC red 610, LC red 610, LC red 640, LCred 670, LC red 705 (Roche Applies Science), Texas red, FAM, TET, HEX,JOE, TMR, and ROX. Quenchers that may be used include, for example,DDQ-I, DDQ-II (Eurogentec), Eclipse (Epoch Biosciences), Iowa Black FQ,Iowa Black RQ (Integrated DNA Technologies), BHQ-1, BHQ-2, BHQ-3(Biosearch Technologies), QSY-7, QSY-21 (Molecular Probes), and Dabcyl.

In some embodiments, a method provided herein may be monitored in anapparatus containing a light source and an optical sensor. In somesituations, the reaction may be positioned in the path of light from thelight source, and light absorbed by the sample (e.g. in the case of aturbid reaction), scattered by the sample (e.g. in the case of a turbidreaction), or emitted by the sample (e.g. in the case of a reactioncontaining a fluorescent molecule) may be measured. In some embodiments,a method provided herein may be performed or monitored in a device ormodule therein as disclosed in U.S. patent application Ser. No.13/769,779, filed Feb. 18, 2013, which is herein incorporated byreference in its entirety.

Systems and Devices

In embodiments, systems and devices provided herein may be deployed inairplanes or airports, in order to test travelers for one or morepathogens, symptoms, or conditions described herein. In embodiments,systems and devices provided herein may be deployed in rural locationsor in locations which are relatively far from a traditional laboratory(e.g. at least 10, 20, 50, 100, 200, or 500 miles from a traditionallaboratory). In embodiments, systems and devices provided herein mayhave one or both of audio and visual input and output. Systems anddevices provided herein with one or both of audio and visualfunctionalities may permit, for example, a subject to participate in atelemedicine session with a healthcare professional located at a remotelocation from the subject through a system or device provided herein. Inembodiments, a healthcare professional may be remotely provided to asubject through a system or device provided herein, wherein thehealthcare professional is the same gender as the subject seeking thehealthcare consultation. For example, a female subject may want to see afemale doctor, but there may be no female doctor near the location ofthe female subject. Through a system or device provided herein havingone or both of audio and visual input and output, the female subject canconsult with a female doctor who is located remotely from the locationof the female subject.

The assays and methods disclosed herein may be performed on a device, oron a system, for processing a sample. The assays and methods disclosedherein can be readily incorporated into and used in an automated assaydevice, and in an automated assay system. For example, systems asdisclosed herein may include a communication assembly for transmittingor receiving a protocol based on the analyte to be detected (e.g., anEbola virus, such as an Ebola Zaire virus, an anti-Ebola antibody, or anelectrolyte) or based on other analytes to be detected by the device orsystem. In embodiments, an assay protocol may be changed based onoptimal scheduling of a plurality of assays to be performed by a device,or may be changed based on results previously obtained from a samplefrom a subject, or based on results previously obtained from a differentsample from the subject. In embodiments, a communication assembly maycomprise a channel for communicating information from said device to acomputer, said wherein said channel is selected from a computer network,a telephone network, a metal communication link, an opticalcommunication link, and a wireless communication link. In embodiments,systems as disclosed herein may transmit signals to a central location,or to an end user, and may include a communication assembly fortransmitting such signals. Systems as disclosed herein may be configuredfor updating a protocol as needed or on a regular basis.

Accordingly, Applicants disclose devices configured to measure ananalyte such as an Ebola virus in a sample of blood according to amethod disclosed herein. Devices configured to measure an analyte suchas, e.g., an Ebola virus, an anti-Ebola antibody, or an electrolyte in asample of blood according to a method disclosed herein may be configuredto detect or measure amounts of an analyte such as, e.g., an Ebolavirus, an anti-Ebola antibody, or an electrolyte from a sample of bloodthat comprises no more than about 1000 μL of blood, or no more thanabout 500 μL of blood, no more than about 250 μL of blood, or no morethan about 150 μL of blood, or no more than about 100 μL of blood, or nomore than about 50 μL of blood, or, in embodiments, wherein said sampleof blood comprises no more than about 25 μL of blood, or wherein saidsample of blood comprises no more than about 10 μL of blood, or whereinsaid sample of blood comprises less than about 10 μL of blood. Suchdevices may be configured to detect or measure an analyte such as, e.g.,an Ebola virus, an anti-Ebola antibody, or an electrolyte in a sample ofblood in less than about three hours, less than about two hours, or lessthan about one hour, or, in embodiments, in less than about 40 minutes,or in less than about 30 minutes.

Devices disclosed herein may be configured to perform an assay for thedetection or measurement of an analyte such as, e.g., an Ebola virus, ananti-Ebola antibody, or an electrolyte, and also to perform an assay forthe measurement of another analyte in the blood sample. Devicesdisclosed herein may be configured to perform an assay for the detectionor measurement of an analyte such as, e.g., an Ebola virus, ananti-Ebola antibody, or an electrolyte, and also to perform an assaycomprising the measurement of a morphological characteristic of a bloodcell in the blood sample. Devices disclosed herein may be configured toperform an assay for the detection or measurement of an analyte, e.g.,an Ebola virus, an anti-Ebola antibody, or an electrolyte and also toperform an assay comprising the measurement of another blood analyte,e.g., a vitamin, a hormone, a drug or metabolite of a drug, or otheranalyte. Such devices may be configured wherein the assays, or the orderof performance of assays, that are performed by said device may bealtered by communication with another device.

Applicants also disclose systems comprising a device as disclosedherein. In embodiments, the system comprises a device that is configuredto perform an assay for the detection or measurement of an analyte,e.g., an Ebola virus, an anti-Ebola antibody, or an electrolyte, andalso to perform an assay for the measurement of another analyte in theblood sample. In embodiments, the system comprises a device that isconfigured to perform an assay for the detection or measurement of ananalyte, such as, e.g., an Ebola virus, an anti-Ebola antibody, or anelectrolyte, and also to perform an assay for the measurement of amorphological characteristic of a blood cell in the blood sample. Inembodiments of such a system, assays, or the order of performance ofassays, that are performed by said device may be altered bycommunication with another device.

Methods and compositions disclosed herein provide rapid assays whichrequire only small amounts of sample, such as only small amounts ofblood. Device and systems disclosed herein are configured to performsuch rapid assays which require only small amounts of sample, such asonly small amounts of blood. Accordingly, the methods, compositions,devices, and systems provide rapid tests, which require only smallbiological samples, and thus provide advantages over other methods,compositions, assays, devices, and systems.

The assays and methods disclosed herein may be performed on a device, oron a system, for processing a sample. The assays and methods disclosedherein can be readily incorporated into and used in device forprocessing a sample, or a system for processing a sample, which may bean automated assay device, or may be an automated assay system. Such adevice, and such a system, may be useful for the practice of the methodsdisclosed herein. For example, a device may be useful for receiving asample. A device may be useful for preparing, or for processing asample. A device may be useful for performing an assay on a sample. Adevice may be useful for obtaining data from a sample. A device may beuseful for transmitting data obtained from a sample. A device may beuseful for disposing of a sample following processing or assaying of asample.

A device may be part of a system, a component of which may be anautomatic assay device. A device may be an automatic assay device. Anautomatic assay device may be configured to facilitate collection of asample, prepare a sample for a clinical test, or effect a chemicalreaction with one or more reagents or other chemical or physicalprocessing, as disclosed herein. An automatic assay device may beconfigured to obtain data from a sample. An automatic assay device maybe configured to transmit data obtained from a sample. An automaticassay device may be configured to analyze data from a sample. Anautomatic assay device may be configured to communicate with anotherdevice, or a laboratory, or an individual affiliated with a laboratory,to analyze data obtained from a sample.

An automatic assay device may be configured to be placed in or on asubject. An automatic assay device may be configured to accept a samplefrom a subject, either directly or indirectly. A sample may be, forexample, a blood sample (e.g., a sample obtained from a fingerstick, orfrom venipuncture, or an arterial blood sample), a urine sample, abiopsy sample, a tissue slice, stool sample, or other biological sample;a water sample, a soil sample, a food sample, an air sample; or othersample. A blood sample may comprise, e.g., whole blood, plasma, orserum. An automatic assay device may receive a sample from the subjectthrough a housing of the device. The sample collection may occur at asample collection site, or elsewhere. The sample may be provided to thedevice at a sample collection site.

In some embodiments, an automatic assay device may be configured toaccept or hold a cartridge. In some embodiments, an automatic assaydevice may comprise a cartridge. The cartridge may be removable from theautomatic assay device. In some embodiments, a sample may be provided tothe cartridge of the automatic assay device. Alternatively, a sample maybe provided to another portion of an automatic assay device. Thecartridge and/or device may comprise a sample collection unit that maybe configured to accept a sample.

A cartridge may include a sample, and may include reagents for use inprocessing or testing a sample, disposables for use in processing ortesting a sample, or other materials. Following placement of a cartridgeon, or insertion of a cartridge into, an automatic assay device, one ormore components of the cartridge may be brought into fluid communicationwith other components of the automatic assay device. For example, if asample is collected at a cartridge, the sample may be transferred toother portions of the automatic assay device. Similarly, if one or morereagents are provided on a cartridge, the reagents may be transferred toother portions of the automatic assay device, or other components of theautomatic assay device may be brought to the reagents. In someembodiments, the reagents or components of a cartridge may remainon-board the cartridge. In some embodiments, no fluidics are includedthat require tubing or that require maintenance (e.g., manual orautomated maintenance).

In embodiments, a cartridge may include reagents for use in nucleic acidassays for processing or testing a sample by detecting or quantifyingnucleic acid targets in the sample, and may also include disposables foruse in processing or testing a sample, or other materials. Inembodiments, a cartridge may include all reagents for use in nucleicacid assays for processing or testing a sample by detecting orquantifying nucleic acid targets in the sample, and may also include alldisposables required for processing or testing a sample.

In embodiments, a cartridge may include reagents for use in amino acidassays (e.g. ELISAs) for processing or testing a sample by detecting orquantifying proteins, peptides, or other proteinaceous material in thesample, and may also include disposables for use in processing ortesting a sample, or other materials. In embodiments, a cartridge mayinclude reagents for use in general chemistry assays for processing ortesting a sample by detecting or quantifying target material (e.g.,vitamins, hormones, metabolites, and other molecules) in the sample Inembodiments, a cartridge may include reagents for use in receptor-basedassays for processing or testing a sample by detecting or quantifyingtarget material in the sample that binds specifically to the receptorsused in the receptor-based assays. In embodiments, a cartridge mayinclude reagents for use in further assays for processing or testing asample by detecting or quantifying electrolytes in the sample. Inembodiments, a cartridge may include reagents for use in further assaysfor processing or testing a sample by detecting or quantifying cells inthe sample by cytometry.

Accordingly, in embodiments, a cartridge may include all reagentsnecessary for performing a nucleic acid assay in an automatic sampleanalysis device or system; and, in embodiments, that cartridge mayfurther carry a sample, may further carry pipettes, mixing vessels,cuvettes, waste containers, or other implements and tools useful for theperformance of such nucleic acid assays. In embodiments, a cartridge mayinclude all reagents necessary for performing an amino acid assay in anautomatic sample analysis device or system; and, in embodiments, thatcartridge may further carry a sample, may further carry pipettes, mixingvessels, cuvettes, waste containers, or other implements and toolsuseful for the performance of such amino acid assays. In embodiments, acartridge may include all reagents necessary for performing a generalchemistry assay in an automatic sample analysis device or system; and,in embodiments, that cartridge may further carry a sample, may furthercarry pipettes, mixing vessels, cuvettes, waste containers, or otherimplements and tools useful for the performance of such generalchemistry acid assays. In embodiments, a cartridge may include allreagents necessary for performing an electrolyte assay in an automaticsample analysis device or system; and, in embodiments, that cartridgemay further carry a sample, may further carry pipettes, mixing vessels,cuvettes, waste containers, or other implements and tools useful for theperformance of such electrolyte assays. In embodiments, a cartridge mayinclude all reagents necessary for performing a receptor-based assay inan automatic sample analysis device or system; and, in embodiments, thatcartridge may further carry a sample, may further carry pipettes, mixingvessels, cuvettes, waste containers, or other implements and toolsuseful for the performance of such receptor-based assays. Inembodiments, a cartridge may include all reagents necessary forperforming a cytometric assay in an automatic sample analysis device orsystem; and, in embodiments, that cartridge may further carry a sample,may further carry pipettes, mixing vessels, cuvettes, waste containers,or other implements and tools useful for the performance of suchcytometric assays. In embodiments, a cartridge may include reagents,tools, implements, or a sample, or combinations thereof, for any two ormore of nucleic acid assays, amino acid assays, general chemistryassays, electrolyte assays, and receptor-based assays.

For example, a cartridge may include reagents for use in processing ortesting a sample for the presence of viral nucleic acids (e.g., EbolaRNA); may include reagents for use in processing or testing a sample forthe presence of viral amino acids (e.g., Ebola nucleoprotein, or otherproteins and proteinaceaous material indicative of Ebola infection); mayinclude reagents for use in processing or testing a sample for thepresence of antibodies to a virus (e.g., antibodies to Ebola virusantigens); may include reagents for use in processing or testing asample for the presence of electrolytes (e.g., for detecting sodium orpotassium level abnormalities in a subject's blood); and combinationsthereof. A cartridge may include vessels, such as, e.g., reagentvessels, mixing vessels, waste vessels, or other vessels, for use inprocessing or testing a sample. A cartridge may include disposables foruse in processing or testing a sample. A cartridge may include cuvettes,and may include other components, elements, and materials for use inprocessing or testing a sample. A cartridge may include at least onesample. Optionally, the cartridge includes at least one or more slotsfor the insertion of one or more containers that may contain one or moresamples.

In embodiments, a sample may be a blood sample, such as a small volumeblood sample, e.g., a fingerstick blood sample. The blood sample may bewhole blood; plasma; serum; heparin-treated whole blood; ethylenediamine tetra-acetic acid (EDTA)-treated whole-blood; centrifuged blood;filtered blood; or other form of blood sample.

A sample or reagent may be transferred to a device, such as an automaticassay device. A sample or reagent may be transferred within a device.Such transfer of sample or reagent may be accomplished without providinga continuous fluid pathway from cartridge to device. Such transfer ofsample or reagent may be accomplished without providing a continuousfluid pathway within a device. In embodiments, such transfer of sampleor reagent may be accomplished by a sample handling system (e.g., apipette); for example, a sample, reagent, or aliquot thereof may beaspirated into an open-tipped transfer component, such as a pipette tip,which may be operably connected to a sample handling system whichtransfers the tip, with the sample, reagent, or aliquot thereofcontained within the tip, to a location on or within the automatic assaydevice. The sample, reagent, or aliquot thereof can be deposited at alocation on or within the automatic assay device. Sample and reagent, ormultiple reagents, may be mixed using a sample handling system in asimilar manner. One or more components of the cartridge may betransferred in an automated fashion to other portions of the automaticassay device, and vice versa.

A device, such as an automatic assay device, may have a fluid handlingsystem. A fluid handling system may perform, or may aid in performing,transport, dilution, extraction, aliquotting, mixing, and other actionswith a fluid, such as a sample. In some embodiments, a fluid handlingsystem may be contained within a device housing. A fluid handling systemmay permit the collection, delivery, processing and/or transport of afluid, dissolution of dry reagents, mixing of liquid and/or dry reagentswith a liquid, as well as collection, delivery, processing and/ortransport of non-fluidic components, samples, or materials. The fluidmay be a sample, a reagent, diluent, wash, dye, or any other fluid thatmay be used by the device, and may include, but not limited to,homogenous fluids, different liquids, emulsions, suspensions, and otherfluids. A fluid handling system, including without limitation a pipette,may also be used to transport vessels (with or without fluid containedtherein) around the device. The fluid handling system may dispense oraspirate a fluid. The sample may include one or more particulate orsolid matter floating within a fluid.

In embodiments, a fluid handling system may comprise a pipette, pipettetip, syringe, capillary, or other component. The fluid handling systemmay have portion with an interior surface and an exterior surface and anopen end. The fluid handling system may comprise a pipette, which mayinclude a pipette body and a pipette nozzle, and may comprise a pipettetip. A pipette tip may or may not be removable from a pipette nozzle. Inembodiments, a fluid handling system may use a pipette mated with apipette tip; a pipette tip may be disposable. A tip may form afluid-tight seal when mated with a pipette. A pipette tip may be usedonce, twice, or more times. In embodiments, a fluid handling system mayuse a pipette or similar device, with or without a pipette tip, toaspirate, dispense, mix, transport, or otherwise handle the fluid. Thefluid may be dispensed from the fluid handling system when desired. Thefluid may be contained within a pipette tip prior to being dispensed,e.g., from an orifice in the pipette tip. In embodiments, or instancesduring use, all of the fluid may be dispensed; in other embodiments, orinstances during use, a portion of the fluid within a tip may bedispensed. A pipette may selectively aspirate a fluid. The pipette mayaspirate a selected amount of fluid. The pipette may be capable ofactuating stirring mechanisms to mix the fluid within the tip or withina vessel. The pipette may incorporate tips or vessels creatingcontinuous flow loops for mixing, including of materials or reagentsthat are in non-liquid form. A pipette tip may also facilitate mixtureby metered delivery of multiple fluids simultaneously or in sequence,such as in 2-part substrate reactions.

The fluid handling system may include one or more fluidically isolatedor hydraulically independent units. For example, the fluid handlingsystem may include one, two, or more pipette tips. The pipette tips maybe configured to accept and confine a fluid. The tips may be fluidicallyisolated from or hydraulically independent of one another. The fluidcontained within each tip may be fluidically isolated or hydraulicallyindependent from one fluids in other tips and from other fluids withinthe device. The fluidically isolated or hydraulically independent unitsmay be movable relative to other portions of the device and/or oneanother. The fluidically isolated or hydraulically independent units maybe individually movable. A fluid handling system may comprise one ormore base or support. A base or support may support one or more pipetteor pipette units. A base or support may connect one or more pipettes ofthe fluid handling system to one another.

An automatic assay device may be configured to perform processing stepsor actions on a sample obtained from a subject. Sample processing mayinclude sample preparation, including, e.g., sample dilution, divisionof a sample into aliquots, extraction, contact with a reagent,filtration, separation, centrifugation, or other preparatory orprocessing action or step. An automatic assay device may be configuredto perform one or more sample preparation action or step on the sample.Optionally, a sample may be prepared for a chemical reaction and/orphysical processing step. A sample preparation action or step mayinclude one or more of the following: centrifugation, separation,filtration, dilution, enriching, purification, precipitation,incubation, pipetting, transport, chromatography, cell lysis, cytometry,pulverization, grinding, activation, ultrasonication, micro columnprocessing, processing with magnetic beads, processing withnanoparticles, or other sample preparation action or steps. For example,sample preparation may include one or more step to separate blood intoserum and/or particulate fractions, or to separate any other sample intovarious components. Sample preparation may include one or more step todilute and/or concentrate a sample, such as a blood sample, or otherbiological samples. Sample preparation may include adding ananti-coagulant or other ingredients to a sample. Sample preparation mayalso include purification of a sample. In embodiments, all sampleprocessing, preparation, or assay actions or steps are performed by asingle device. In embodiments, all sample processing, preparation, orassay actions or steps are performed within a housing of a singledevice. In embodiments, most sample processing, preparation, or assayactions or steps are performed by a single device, and may be performedwithin a housing of a single device. In embodiments, many sampleprocessing, preparation, or assay actions or steps are performed by asingle device, and may be performed within a housing of a single device.In embodiments, sample processing, preparation, or assay actions orsteps may be performed by more than one device.

An automatic assay device may be configured to run one or more assay ona sample, and to obtain data from the sample. An assay may include oneor more physical or chemical treatments, and may include running one ormore chemical or physical reactions. An automatic assay device may beconfigured to perform one, two or more assays on a small sample ofbodily fluid. One or more chemical reaction may take place on a samplehaving a volume, as described elsewhere herein. For example one or morechemical reaction may take place in a pill having less than femtolitervolumes. In an instance, the sample collection unit is configured toreceive a volume of the bodily fluid sample equivalent to a single dropor less of blood or interstitial fluid. In embodiments, the volume of asample may be a small volume, where a small volume may be a volume thatis less than about 1000 μL, or less than about 500 μL, or less thanabout 250 μL, or less than about 150 μL, or less than about 100 μL, orless than about 75 μL, or less than about 50 μL, or less than about 40μL, or less than about 20 μL, or less than about 10 μL, or other smallvolume. In embodiments, all sample assay actions or steps are performedon a single sample. In embodiments, all sample assay actions or stepsare performed by a single device. In embodiments, all sample assayactions or steps are performed within a housing of a single device. Inembodiments, most sample assay actions or steps are performed by asingle device, and may be performed within a housing of a single device.In embodiments, many sample assay actions or steps are performed by asingle device, and may be performed within a housing of a single device.In embodiments, sample processing, preparation, or assay actions orsteps may be performed by more than one device.

An automatic assay device may be configured to perform a plurality ofassays on a sample. In embodiments, an automatic assay device may beconfigured to perform a plurality of assays on a single sample. Inembodiments, an automatic assay device may be configured to perform aplurality of assays on a single sample, where the sample is a smallsample. For example, a small sample may have a sample volume that is asmall volume of less than about 1000 μL, or less than about 500 μL, orless than about 250 μL, or less than about 150 μL, or less than about100 μL, or less than about 75 μL, or less than about 50 μL, or less thanabout 40 μL, or less than about 20 μL, or less than about 10 μL, orother small volume. An automatic assay device may be capable ofperforming multiplexed assays on a single sample. A plurality of assaysmay be run simultaneously; may be run sequentially; or some assays maybe run simultaneously while others are run sequentially. One or morecontrol assays and/or calibrators (e.g., including a configuration witha control of a calibrator for the assay/tests) can also be incorporatedinto the device; control assays and assay on calibrators may beperformed simultaneously with assays performed on a sample, or may beperformed before or after assays performed on a sample, or anycombination thereof. In embodiments, all sample assay actions or stepsare performed by a single device. In embodiments, all of a plurality ofassay actions or steps are performed within a housing of a singledevice. In embodiments, most sample assay actions or steps, of aplurality of assays, are performed by a single device, and may beperformed within a housing of a single device. In embodiments, manysample assay actions or steps, of a plurality of assays, are performedby a single device, and may be performed within a housing of a singledevice. In embodiments, sample processing, preparation, or assay actionsor steps may be performed by more than one device.

In embodiments, all of a plurality of assays may be performed in a shorttime period. In embodiments, such a short time period comprises lessthan about three hours, or less than about two hours, or less than aboutone hour, or less than about 40 minutes, or less than about 30 minutes,or less than about 25 minutes, or less than about 20 minutes, or lessthan about 15 minutes, or less than about 10 minutes, or less than about5 minutes, or less than about 4 minutes, or less than about 3 minutes,or less than about 2 minutes, or less than about 1 minute, or othershort time period.

An automatic assay device may perform nucleic acid assays, includingisothermal nucleic acid assays (e.g., assays for detecting and measuringnucleic acid targets in a sample, including DNA and RNA targets). Inembodiments, an automatic assay device may perform nucleic acid assaysas disclosed in U.S. patent application Ser. No. 14/183,503, filed Feb.18, 2014; U.S. patent application Ser. No. 14/214,850, filed Mar. 15,2014; International Patent Application PCT/US2014/030034, filed Mar. 15,2014; and in International Patent Application PCT/US2014/056151, filedSep. 17, 2014. An automatic assay device may perform antibody assays,including enzyme-linked immunosorbent assays (ELISA), and other assaysfor detecting and measuring the amounts of proteins (includingantibodies), peptides, and small molecules in samples. An automaticassay device may perform general chemistry assays, including electrolyteassays (e.g., assays for detecting and measuring the amounts ofelectrolytes such as sodium and potassium in a sample).

An automatic assay device may be configured to detect one or moresignals relating to the sample. An automatic assay device may beconfigured to identify one or more properties of the sample. Forinstance, the automatic assay device may be configured to detect thepresence or concentration of one analyte or a plurality of analytes or adisease condition in the sample (e.g., in or through a bodily fluid,secretion, tissue, or other sample). Alternatively, the automatic assaydevice may be configured to detect a signal or signals that may beanalyzed to detect the presence or concentration of one or more analytes(which may be indicative of a disease condition) or a disease conditionin the sample. The signals may be analyzed on board the device, or atanother location. Running a clinical test may or may not include anyanalysis or comparison of data collected.

A chemical reaction or other processing step may be performed, with orwithout the sample. Examples of steps, tests, or assays that may beprepared or run by the device may include, but are not limited toimmunoassay, nucleic acid assay, receptor-based assay, cytometric assay,colorimetric assay, enzymatic assay, electrophoretic assay,electrochemical assay, spectroscopic assay, chromatographic assay,microscopic assay, topographic assay, calorimetric assay, turbidmetricassay, agglutination assay, radioisotope assay, viscometric assay,coagulation assay, clotting time assay, protein synthesis assay,histological assay, culture assay, osmolarity assay, and/or other typesof assays, centrifugation, separation, filtration, dilution, enriching,purification, precipitation, pulverization, incubation, pipetting,transport, cell lysis, or other sample preparation action or steps, orcombinations thereof. Steps, tests, or assays that may be prepared orrun by the device may include imaging, including microscopy, cytometry,and other techniques preparing or utilizing images. Steps, tests, orassays that may be prepared or run by the device may further include anassessment of histology, morphology, kinematics, dynamics, and/or stateof a sample, which may include such assessment for cells.

A device may be capable of performing all on-board steps (e.g., steps oractions performed by a single device) in a short amount of time. Adevice may be capable of performing all on-board steps on a singlesample in a short amount of time. For example, from sample collectionfrom a subject to transmitting data and/or to analysis may take about 3hours or less, 2 hours or less, 1 hour or less, 50 minutes or less, 45minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes orless, 15 minutes or less, 10 minutes or less, 5 minutes or less, 4minutes or less, 3 minutes or less, 2 minutes or less, or 1 minute orless. The amount of time from accepting a sample within the device totransmitting data and/or to analysis from the device regarding such asample may depend on the type or number of steps, tests, or assaysperformed on the sample. The amount of time from accepting a samplewithin the device to transmitting data and/or to analysis from thedevice regarding such a sample may take about 3 hours or less, 2 hoursor less, 1 hour or less, 50 minutes or less, 45 minutes or less, 40minutes or less, 30 minutes or less, 20 minutes or less, 15 minutes orless, 10 minutes or less, 5 minutes or less, 4 minutes or less, 3minutes or less, 2 minutes or less, or 1 minute or less.

A device may be configured to prepare a sample for disposal, or todispose of a sample, such as a biological sample, following processingor assaying of a sample.

In embodiments, an automatic assay device may be configured to transmitdata obtained from a sample. In embodiments, an automatic assay devicemay be configured to communicate over a network. An automatic assaydevice may include a communication module that may interface with thenetwork. An automatic assay device may be connected to the network via awired connection or wirelessly. The network may be a local area network(LAN) or a wide area network (WAN) such as the Internet. In someembodiments, the network may be a personal area network. The network mayinclude the cloud. The automatic assay device may be connected to thenetwork without requiring an intermediary device, or an intermediarydevice may be required to connect an automatic assay device to anetwork. An automatic assay device may communicate over a network withanother device, which may be any type of networked device, including butnot limited to a personal computer, server computer, or laptop computer;personal digital assistants (PDAs) such as a Windows CE device; phonessuch as cellular phones, smartphones (e.g., iPhone, Android, Blackberry,etc.), or location-aware portable phones (such as GPS); a roamingdevice, such as a network-connected roaming device; a wireless devicesuch as a wireless email device or other device capable of communicatingwireless with a computer network; or any other type of network devicethat may communicate possibly over a network and handle electronictransactions. Such communication may include providing data to a cloudcomputing infrastructure or any other type of data storageinfrastructure which may be accessed by other devices.

An automatic assay device may provide data regarding a sample to, e.g.,a health care professional, a health care professional location, such asa laboratory, or an affiliate thereof. One or more of a laboratory,health care professional, or subject may have a network device able toreceive or access data provided by the automatic assay device. Anautomatic assay device may be configured to provide data regarding asample to a database. An automatic assay device may be configured toprovide data regarding a sample to an electronic medical records system,to a laboratory information system, to a laboratory automation system,or other system or software. An automatic assay device may provide datain the form of a report.

A laboratory, device, or other entity or software may perform analysison data regarding a sample in real-time. A software system may performchemical analysis and/or pathological analysis, or these could bedistributed amongst combinations of lab, clinical, and specialty orexpert personnel. Analysis may include qualitative and/or quantitativeevaluation of a sample. Data analysis may include a subsequentqualitative and/or quantitative evaluation of a sample. Optionally, areport may be generated based on raw data, pre-processed data, oranalyzed data. Such a report may be prepared so as to maintainconfidentiality of the data obtained from the sample, the identity andother information regarding the subject from whom a sample was obtained,analysis of the data, and other confidential information. The reportand/or the data may be transmitted to a health care professional. Dataobtained by an automatic assay device, or analysis of such data, orreports, may be provided to a database, an electronic medical recordssystem, to a laboratory information system (LIS), to a laboratoryautomation system (LAS), or other system or software.

In one embodiment, nucleic acid amplification (NAA) is performed from acapillary blood sample. The nucleic acid amplification may bequalitative or quantitative. In one embodiment, the portion of thesample for NAA is processed to remove formed components and leave onlyplasma. Optionally, the portion of the sample for NAA is processed toremove formed components and leave only serum. Optionally, the portionof the sample for NAA comprises capillary blood that is processed withat least one pre-treatment such as but not limited to at least oneanti-coagulant compatible with the NAA technique.

In one non-limiting example, the collection of the sample may be by wayof a sample collection device such as but not limited to those describedin PCT Patent Application Ser. No. PCT/US14/30792 filed Mar. 17, 2014and PCT Patent Application Ser. No. PCT/US13/00268 filed Dec. 5, 2013,both fully incorporated herein by reference for all purposes.

In another non-limiting example, the process may involve shipping and/orcollecting fingerstick samples of capillary blood and/or otherbiological sample for analysis, wherein such analysis may include but isnot limited to nucleic acid amplification, electrolyte(s) measurement,and combined antibody measurement from the same original sample. Also,some embodiments may include complete blood count (CBC) and cellcounting for infection triage and management, all on the same samplecollected from the subject at one collection.

In one non-limiting example, the process may involve use of a phenolbased inactivation material on the sample to inactivate a virus, such asthe Ebola virus (e.g., Ebola Zaire virus), wherein such inactivationmaterial may be but is not limited to Trizol (Invitrogen) (comprising byweight: 30-60% Phenol, 15-40% Guanidine isothiocyanate, and 7-13%Ammonium thiocyanate) or other inactivation agent in sample collectionvial(s), sample collection circuit, or the like to fully deactivatesample for transport or prior to instrument use. Optionally, someembodiments may use RNeasy (Qiagen) and/or Tripure (Roche) reagent(s).In one non-limiting example, sample inactivation is accomplished throughuse of a one-step sample homogenization/lysis procedure, in which theTriPure isolation reagent disrupts cells and denatures endogenousnucleases, wherein chloroform may also be added along with or after theTriPure reagent. Other optional virus inactivation techniques mayinclude use of: bleach such as but not limited to 1:10 Bleach solution,5% Lysol/phenolics, micro-chem/quaternary ammonium,incineration/autoclave, and or virus labile to desiccation and UV. Someof these inactivation techniques may be applied to sample after thesample has been processed for analyte levels. Optionally, some may beapplied to certain portion(s) of the sample, such as a pellet of formedblood components, that will not be analyzed. Some embodiments may havean SPU with inactivation to be conducted in the device, wherein the userloads a sample container with active sample into a cartridge and insertsthe cartridge into the SPU. In such an embodiment, the SPU willautomatically handle inactivation to all or parts of the sample attime(s) as determined by programmable protocol being executed by theSPU. Some embodiments may include inactivation material in the cartridgeC. Optionally, some embodiment may contain inactivation material in areservoir that is onboard the SPU and not part of the cartridge C.Optionally, some embodiments may allow for insertion of two cartridgesinto the SPU, which in one non-limiting example, one cartridge hasreagents and one cartridge has inactivation material(s).

Alternatively, the process may inactivate the sample through lysis andextraction steps in device (or both). Optionally, some embodiments mayuse an activated sample so long as a cap or cover is added prior tovigorous motion. Optionally, still further embodiments may centrifugethe collection vial prior to insertion into the device, or optionally,centrifuge inside the device. Optionally, other formed componentseparation techniques may be used on the sample prior to insertion intothe device, or optionally, inside the device. Optionally, someembodiments may use a further containment enclosure around the device tominimize the risk of contamination outside of the device.

An automatic sample analysis device may have a housing; such a housingmay act as a containment enclosure. In embodiments, an automatic sampleanalysis device may be placed within an enclosure, such as a containmentenclosure. An automatic sample analysis device placed within anenclosure may have a housing, and in such an embodiment the housing thuswill be enclosed within the enclosure.

A housing may include one or more air filters, such as, e.g., highefficiency particulate air (HEPA) filters suitable for blocking the flowof contaminants and micro-organisms. A containment enclosure may includeone or more air filters, such as, e.g., HEPA filters suitable forblocking the flow of contaminants and micro-organisms. In embodiments,air filters may filter outside air as it flows to, or as it enters, orboth, the interior of the containment enclosure, effective to reduce orprevent the entry of dust or other contamination into the SPU, to reduceor prevent interference with the operation of the SPU, and to reduce orprevent damage to the SPU. In embodiments, air filters may filter air asit flows outwardly, or as it leaves, or both, the interior of thecontainment enclosure, effective to reduce or prevent contamination(whether droplets, particulate matter, or other contaminants) fromexiting the SPU, effective to reduce or prevent transport or spread ofhazardous material out of the SPU. In embodiments, an air filtersuitable for use in a containment enclosure is capable of filtering outthe vast majority of particles (i.e., preventing the passage of theparticles through the filter) in air entering the filter. For example, asuitable air filter may filter out greater than about 98% of the 0.3micron (0.3 μm) diameter particles in air entering the filter. Inembodiments, a suitable air filter may filter out greater than about 99%of the 0.3 μm diameter particles in air entering the filter; or greaterthan about 99.5%, or greater than about 99.7%, or greater than about99.8%, or greater than about 99.9%, or greater than about 99.95%, orgreater than about 99.995%, or greater than about 99.9995%, or greaterthan about 99.99995%, or greater than about 99.999995%, or greater, ofthe 0.3 μm diameter particles in air entering the filter.

In one non-limiting example, sensitive (low limit of detection [LoD])NAA is performed on capillary samples from dilutions orpre-amplification thermal cycling to increase sensitivity (especially onan isothermal assay). Additionally, some embodiments may use anisothermal assay from capillary samples to generate real-time data (<1hour and often <30 minutes on positive patients) for real-time triageand to be able to effect quarantine on subject(s) that test positive.

Optionally, some embodiments may use a sample collection device withformed component separation capability for obtaining plasma or otherwiseseparating out cells, such as but not limited to a sample collectiondevice described in PCT Patent Application Ser. No. PCT/US14/30070 filedMar. 15, 2014 and fully incorporated herein by reference for allpurposes. Optionally, the system may provide any combination of testsrun from a fingerstick sample shipped as whole blood or shipped andseparated into plasma immediately after collection through a collectioncircuit.

Embodiments of the system may provide the ability to put the system intohot zones themselves as opposed to reference labs to facilitatequarantine on the spot without wait times for tests to be couriered off,and the therefore facilitate containment, wherein active samples are notbeing transported outside the hot zone.

Also, some embodiments may use customized trucks with sample processingdevices on them with optionally, one or more of the following: powerbuilt in/generators, refrigerators, connectivity including satelliteconnectivity, and/or security for driving/locating in disaster zones.These trucks or similar vehicles can distribute and create decentralizedlocations around which to do real-time quarantine for containment.Optionally, some embodiment may use trucks or mobile units for Eboladetection that is decentralized to distributed locations with built inpower and satellite connectivity for real-time triage and quarantine,with containment housing structures with beds built around them.

In embodiments, an SPU may be a modular device used for performingpre-analytic functions (e.g., one or more of sample dilution, samplealiquotting, sample preparation, and other functions) as describedherein. An SPU may be designed to automatically replicate the processingsystems used in the relevant traditional, manual or partially manual,assay protocols. In embodiments, an SPU may be enclosed in a thermallyinsulated and light-tight sheet metal enclosure. In embodiments, an SPUmay have some, and in embodiments, may have all, of the followingcomponents: Liquid Handling Module, Centrifuge Module, Sonicator Module,Magnet Tool, one or more detectors (which may include a LuminometerModule, a Fluorometer Module, a Fluorometer/Turbidimeter Module, aSpectrophotometer Module, and a Microscopy Module; in embodiments, anSPU will include all such detectors), a Thermal Control System, and aMachine Vision System. In embodiments, an SPU may be made from anysuitable components, including purchased components, machined parts, andmolded parts. In embodiments, such machined parts may be made ofaluminum, stainless steel, or other metals.

A sample, consumables, reagents, reagent vessels, and other items may beprovided to a SPU via a cartridge. For example, the sample and productsof further processing and reaction may be contained in disposableconsumables inside the disposable reagent tray or Cartridge. Allconsumables are discrete such that reagents and reactions for each assayreside and occur, respectively, in physically separate locations toprevent cross-reactivity. The consumables contain all liquids orreagents such that no sample or reagent ever directly interacts with anSPU. All consumables for processing are contained in the Cartridge (andare not built into the SPU) and may be placed back into the Cartridge atthe completion of processing for disposal.

Consumables which may be used for the NAA assays include, withoutlimitation, the following: Round vessels (e.g., 60 μL capacitypolypropylene vessels for storing reagents, dilutions, mixing, andreactions); Wash vessels (e.g., 200 μL capacity polypropylene vesselsfor storing wash buffers); Centrifuge vessels (e.g., in one embodiment,a narrow diameter 100 μL capacity polystyrene vessels for centrifugingblood and efficiently removing supernatant; or, in another embodiment, a120 μL capacity polypropylene vessels for centrifuging samples andefficiently mixing and transferring small volumes); Mini tips (e.g., 10μL capacity polypropylene tips for transporting fluids; may includesilica filters for preventing cross-contamination); Large tips (e.g., 40μL capacity polypropylene tips for transporting fluids; with silicafilters for preventing cross-contamination); Dynamic Dilution tips(e.g., 10 μL capacity coated polystyrene tips for transportingsolutions; with silica filters for preventing cross-contamination); NAAvessels (e.g., 60 μL capacity polypropylene vessels which serve asreaction vessels for the amplification reaction). In embodiments, afinal fluorescence signal from the product generated in these vesselsmay be detected from NAA vessels.

Further consumables may include, for example, NAA trays (e.g., trayswhich hold multiple NAA vessels, such as 8 NAA vessels); such trays maybe designed so as to be able to be picked up by the fluid handlingmodule to transport the vessels between the Cartridge and the NAA moduleof a SPU. Further consumables may include, for example, a sonicatorvessel (e.g., a 350 μL polystyrene vessel which may be used to containsample during sonication). Further consumables may include, for example,a Magnet Tool Sleeve (e.g., a disposable polypropylene sleeve used toseparate a magnet from a consumable, or sample, or other material orobject, to prevent contamination).

A cartridge may house some or all of the consumables listed above. Acartridge may house a sample and some or all of the consumables listedabove. A cartridge may include a lid to hold all consumables in placeand prevent user interaction. In embodiments, a cartridge may beprovided with a closed lid, under which all consumables required for NAAassays may be carried as required. Reagents and buffers required forperformance of NAA assays may be pre-filled and sealed in vessels (e.g.,in Round vessels and Wash vessels). Similarly, consumables and reagentsfor another type of assay, such as cytometry, ELISA, general chemistry,or other assay, may be provided in a Cartridge. Similarly, consumablesand reagents for other types of assays, such as cytometry, ELISA,general chemistry, and other assays, may be included with those for NAAor with those for other assays in the same Cartridge. Thus, inembodiments, consumables and reagents for other types of assays, such ascytometry and other general chemistry assays, may be included with aCartridge dedicated to a single type of assay, or dedicated to two typesof assays (e.g., two of NAA, Cytometry, ELISA, and general chemistry).In embodiments, consumables and reagents for other types of assays, suchas cytometry and other general chemistry assays, may be included with aCartridge dedicated to three types of assays (e.g., three of NAA,Cytometry, ELISA, and general chemistry), or may include consumables forall of these types of assays. The NAA vessels may be provided pre-filledwith the master mix for the assay, and may include by a protective waxlayer on top of the NAA vessels to contain the master mix.

In embodiments, a Laboratory Automation System (LAS) may include atleast one server configured to communicate with and control one or moreSPUs. For example, communication with and control of one or more SPUsmay be accomplished using an encrypted, certificate-based securitysystem. An LAS may provide a number of functions, includingcommunicating test protocols to the SPU based on the desired tests to berun on the sample and for maintaining oversight over the SPUs. Duringprocessing, the SPU and LAS may communicate to validate the quality andintegrity of the consumables, based on lot information tracked in theLAS, execute the sample processing steps, and monitor and oversee thequality of the sample processing, and perform other functions andoperations. After controlling sample processing in the SPU, signal setsfrom the sample may be transferred to the LAS where the raw data may beanalyzed, the relevant reportables generated for a LaboratoryInformation System, and post-analytic processing steps performed.

In embodiments, an LAS may be overseen by a Clinical LaboratoryImprovements Act (CLIA)-certified laboratory; such oversight may provideoversight and remote control of the SPU. For example, consumablescontaining patient samples (e.g., a Theranos Nanotainer™ tube for bloodsample) may be placed in a Cartridge and may be inserted into the SPU.The SPU may scan a barcode on the Cartridge, and the barcode value maybe communicated to the LAS. The LAS may securely de-code the barcodevalue, and may communicates a sample processing protocol to theprocessor in the SPU. A processor may further distribute tasks receivedfrom the LAS to various modules in the SPU. The SPU may feed informationback to the LAS for monitoring of the SPU and its performance. Inembodiments, the SPU may constantly feed information back to the LAS toensure constant monitoring of the SPU and of its performance. Inembodiments, final steps of sample processing may include signalgeneration (such as, e.g., fluorescence light for NAA assays,chemiluminescence light for ELISA assays, and transmitted lightintensity spectrum for electrolyte assays), and signal detection bydetectors. Such data may be transmitted to the LAS, which may performanalysis on these raw data and yield clinically relevant analytereportables. In embodiments, such analysis performed on these raw data,and such clinically relevant analyte reportables yielded by suchanalysis, may be provided to, and may be useful for, CLIA laboratorystaff to oversee and further analyze, as applicable.

In embodiments, as SPU may operate under the control of an LAS. Forexample, an SPU may be connected to the LAS via a secure Internet orother data network connection, e.g., via Iridium satellite technologyand service. An SPU and an LAS may be connected via two-waycommunication with each other. For example, an LAS can send variouscommands and protocols to the processor of an SPU, for execution by theSPU. Similarly, an SPU can send information obtained by the SPU to anLAS, such as data obtained from pre-analytic steps with a sample orinformation obtained from sensors within an SPU (e.g. signal, image,temperature information). Information sent by an SPU to an LAS may be inresponse to a specific request for information from the LAS to the SPU,or it may be part of a standardized protocol. For example, uponcompletion of pre-analytic processing in the SPU, an LAS may performanalysis and post-analytic processing.

Although a SPU may be situated at a Field Site location which may bephysically separate from an LAS, in embodiments complete control andoversight may be extended from the central LAS to the remote SPU toensure CLIA-oversight and certification of the tests being reported. TheSPU may serve as part of a CLIA-certified laboratory, and laboratoryresults generated from data analyzed in the LAS and obtained from asample processed on a SPU may be CLIA-certified.

In embodiments, a touch screen may be provided in or on an SPU foroperation of the device. Such a touchscreen may allow for detailed,user-oriented instructions, oversight, e.g., by ensuring a technicianfollows all appropriate steps before processing a sample, and two waycommunications. In embodiments, operation of an SPU at a remote locationmay be performed by a phlebotomist or other appropriately authorizedtechnician trained in the operation of an SPU, e.g., trained in theoperation of an SPU at a Field Site location.

In embodiments, an LAS may allow the operation of the clinicallaboratory process without operator intervention. For example, suchoperation without operator intervention may include control of the SPUthrough one or more of direct LAS interfacing, specimen manipulation,transportation of the specimen and related signals, result evaluation,repeat testing, reflex testing and quality assessment and resultsreporting.

In embodiments, a secure communications infrastructure may be used toallow for CLIA-compliance for certified analysis and testing through theLAS for determination of the presence or absence of various substancesin subjects (e.g., in a human subject) in a CLIA-certified laboratorywhile automating sample processing in field through the SPU in FieldSites to minimize pre-analytic error and variability.

Example 1

The samples analyzed in this example were analyzed by an automaticsample analysis device, the SPU. The methods illustrated in this examplemay be applied to other automatic sample analysis devices havingcapabilities as disclosed herein.

An automatic sample processing device or system, such as an SPU, mayinclude fluid handling and fluid transport capabilities, includingsample vessels, mixing vessels, and other containers for holding andprocessing samples; may include a centrifuge, vessels for use with acentrifuge, and other elements or components for use with a centrifuge;may include a sonicator, vessels for use with a sonicator, and otherelements or components for use with a sonicator; may include magnets andmagnetic components for use, e.g., in magnetic separation of componentsof a sample; may include an optical detector, an electronic detectors;may include a light source, a filter, a mirror, a prism, and otheroptical elements and components; and may include other components andcapabilities.

A sample may be provided to (e.g., loaded in) an automatic sampleprocessing device or system, such as an SPU, via a cartridge. Acartridge for use with an automatic sample processing device or system,such as an SPU, may include reagents for sample analysis, vessels foruse in analyzing a sample, and other components, tools, devices, orconsumables for use in analyzing a sample. Thus, a cartridge may carry asample, and be used to provide a sample to an automatic sampleprocessing device or system, such as an SPU. A cartridge may carryreagents and other materials for use in analyzing a sample. Inembodiments, a cartridge may carry a sample and may carry reagents andother materials together with that sample, for use in analyzing thatsample by an automatic sample processing device or system, such as anSPU.

In this example, small volume blood samples are tested for the presenceof Ebola virus markers, and for electrolyte levels indicative of Ebolainfection.

The Sample used for TNAA assay: ethylene diamine tetra acetic acid(EDTA)-anti-coagulated whole blood collected by fingerstick.

The Sample used for ELISA assay: heparin-anticoagulated plasma collectedby a fingerstick as whole blood (processed in the SPU into plasma).EDTA-anti-coagulated whole blood may also be used for the ELISA assay.

The Sample used for Electrolyte assay: heparin-anticoagulated plasmacollected by a fingerstick as whole blood (processed in the SPU intoplasma).

What was measured: Specific nucleic acid sequences from the genome ofthe Ebola Zaire Virus, IgM and IgG antibodies to the Ebola virus, andsodium and potassium levels.

The final steps of sample processing are signal generation (which isfluorescence light for the TNAA assays, chemiluminescence light in thecase of the ELISA assays, and transmitted light intensity spectrum forthe Electrolyte assays).

For the first time, healthcare providers may be able to run a molecularassay for the rapid detection of the Ebola virus at the same time asassays for the antibodies to the Ebola virus along with essentialchemistry assays for sodium and potassium assays to manage electrolyteimbalances in the field where patients are presenting with signs andsymptoms of Ebola infection without requiring transport of samples to aclinical laboratory for testing. In addition, samples may be able to becollected by capillary means, with single use fully retractable lancets,without requiring the needles and trained personnel needed forperforming phlebotomy. Furthermore, systems can be deployed into the hotzone or treatment units where assessment and quarantine can befacilitated in real-time through rapid test run times. The systemfurther facilitates real-time reporting and modeling of cases throughreal-time connectivity to self-learning epidemiological models/softwaresystems, as the assays are run on a system as provided herein forcapillary samples in configurations that enable remote connectivitythrough satellite (so as to not require the presence of cellularnetworks) and distribution to the places at which patients are beingtreated and would ideally be tested and/or quarantined.

In embodiments, further assays to simultaneously measure pathogenscausing other fevers of unknown origin may also be performed, as well asmolecular tests for the other strains of Ebola virus, and hemoglobin andiron tests for anemia. The fevers of unknown origin tests will targetthese conditions: Lassa fever, malaria, and cholera, (ultimately to befollowed by typhoid fever, Marburg, dengue I, II, III and IV,chikungunya, West Nile, and meningitis), with the objective of allowingfor better engagement in the screening process through the inclusion oftests associated with fever like symptoms, so that patients may be lessafraid to present themselves for testing. The anemia tests are includedto facilitate better triaging of patients in the treatment units,focusing on the fact that those infected often have hookworm or otherconditions and die of anemia before being able to get treated for theinfection itself.

The present methods, devices, systems, and kits provide advantagesotherwise not available to detect infectious diseases, and provideimproved methods for treating and for preventing the spread ofinfectious diseases. For example, a real-time diagnostic (RDT) asdisclosed herein allows triage of patients at the entrance of everytreatment unit. This enables the rapid separation of those patients withEbola from other patients who have more typical tropical infections suchas malaria, typhoid, and shigella infections. This reduces the verylarge risk of these other patients being exposed to Ebola infection onthe suspect side of the treatment unit.

In addition, health care workers are commonly infected when theyunsuspectingly tend to an Ebola patient without proper personalprotective equipment (PPE). Additionally these Ebola patients in newoutbreaks often wait in common waiting areas with other people seekingcare and are placed on the ward with other non-ebola patients in thehospital. This practice leads to the avoidable exposure of many othersto infection. These preventable infections could be drasticallycurtailed with the availability of a rapid diagnostic test for Ebola asdisclosed herein.

The methods, devices, systems, and kits disclosed herein provide meansto assess the true distribution and prevalence of Ebola infection withingiven geographic areas at any given point in time, allowing planning forclinical and logistical support. Better data on the spread of theinfection will enable enhanced forecasting of the rates of spread andenabling the focus of awareness training in areas where it would be mosteffective.

FIG. 1 provides an outline of an exemplary system as disclosed herein. Asystem for performing analysis of small-volume blood samples in shortperiods of time may include pre-analytical (sample processing, performedby the SPU as controlled by the LAS), analytical (report generation,performed by the LAS), and post-analytical (report transmission,performed by the LAS) parts.

An exemplary system as described herein comprises the followingcomponents operating under oversight of the Clinical LaboratoryImprovement Amendments (CLIA)-certified laboratory: the SampleProcessing Unit (“SPU”), designed to be deployed to sites where thepatients suspected of infection with the Ebola virus will present fordiagnosis and treatment (“Field Site”), and a centralized LaboratoryAutomation System (“LAS”), which is overseen by the CLIA-certifiedlaboratory, running the assays as disclosed herein. Optionally, itshould be understood that other embodiments may use one or more otherlaboratories certified by an authorized body other than CLIA.

A Field Site may be a field location used to diagnose and treat subjectsfor Ebola virus. Such a site is considered “hot” as potentiallyinfectious Ebola samples are being collected, handled, and disposed. Assuch, those individuals collecting and handling these specimens mustadhere to applicable safety protocols for handing samples presumed to bepositive for Ebola virus. The SPU may be used in this environment, andaccordingly may be assumed to be contaminated. Individuals using the SPUmust use appropriate PPE as required by the Field Site. In embodiments,an SPU may be contained within a protective container, or sheath, orwrapped in plastic or other material in order to reduce or prevent itscontamination, or in order to reduce or prevent contamination of itsouter surface.

In embodiments, primary applications of the TNAA and ELISA assays are(i) to detect the presence of Ebola Virus RNA and (ii) to detect EbolaIgM and IgG in patients that are symptomatic or at risk for viral Ebolainfection, respectively. The primary applications of the Electrolyteassays are to accurately and precisely quantify the concentrations ofSodium and Potassium in patients presenting with signs and symptomsconsistent with infection with Ebola virus.

The sample type for Ebola Zaire assays disclosed herein isEDTA-anti-coagulated whole blood collected by fingerstick. The sampletype for the ELISA and Electrolyte assays is heparin-anticoagulatedplasma collected by a fingerstick as whole blood (processed in the SPUinto plasma). EDTA-anti-coagulated and heparin anti-coagulated, asapplicable, whole blood samples are collected and introduced into adisposable Cartridge at the Field Site and fed to the SPU, where thesamples undergo processing and reaction steps, and are eventuallyintroduced to a detector to yield a set of signals. These signal setsare transferred to the LAS where the raw data are processed and analyzedand oversight is provided, and the relevant reportables (which mayinclude, e.g., raw data, analyzed data, and data plots) are generated.

FIG. 1 shows a schematic diagram of the workflow of the system. Stepsillustrated by boxes numbered from 1 to 4 represent pre-analytic steps.Pre-analytic steps include sample collection, sample processing, reagentaddition, signal generation, and transmission. Steps illustrated byboxes numbered from 5 to 8 represent analytic steps. Analytic stepsinclude analysis of data received from a device at a sample collectionsite, oversight, including analysis of controls, calibrations,replicates, outliers, device and sample identification and qualityinformation, and generation of the reportable. Transmission of thereport to the health care professional represents a post-analytic step.Post-analytic steps include further review of the analysis of data, andreview of report generation and of the report generated for a particulartest prior to sign off by CLIA-laboratory personnel and transmission tothe care provider who ordered a given test.

For field deployment where access to the internet is not available viawired connectivity, connectivity to a Laboratory Automation System maybe maintained via Iridium satellite technology and service. Test resultscan be configured to be displayed on a Sample Processing Unit thatprocessed the sample, and/or results can be automatically uploaded to ahosted cloud for display to authorized care providers. The systemdescribed herein consists of the following components operating underoversight of the Clinical Laboratory Improvement Amendments(CLIA)-certified laboratory: the Sample Processing Unit (“SPU”),designed to be deployed to sites where the patients suspected ofinfection with the Ebola virus will present for diagnosis and treatment(“Field Site”), and a centralized Laboratory Automation System (“LAS”),which is overseen by the CLIA-certified laboratory, running the assaysas disclosed herein.

As shown in the schematic outline of FIG. 1, operation of systems asdisclosed herein may follow the schematic outline of the work-flow ofmethods disclosed herein. In FIG. 1, steps illustrated by boxes numberedfrom 1 to 4 represent pre-analytic steps. Pre-analytic steps includesample collection, sample processing, reagent addition, signalgeneration, and transmission. Steps illustrated by boxes numbered from 5to 8 represent analytic steps. Analytic steps include analysis of datareceived from a device at a sample collection site, oversight, includinganalysis of controls, calibrations, replicates, outliers, device andsample identification and quality information, and generation of thereportable. Transmission of the report to the health care professionalrepresents a post-analytic step. Post-analytic steps include furtherreview of the analysis of data, and review of report generation and ofthe report generated for a particular test prior to sign off byCLIA-laboratory personnel and transmission to the care provider whoordered a given test.

Terms used in FIG. 1 and elsewhere herein include the following.

CLIA-certified laboratory: the Sample Processing Unit (“SPU”), designedto be deployed to sites where the patients suspected of infection withthe Ebola virus will present for diagnosis and treatment (“Field Site”),and a centralized Laboratory Automation System (“LAS”), which isoverseen by the CLIA-certified laboratory, running the assays asdisclosed herein.

Field Site is field location used to diagnose and treat subjects forEbola virus. The SPU may be used in this environment, and accordinglymay be assumed contaminated.

The primary applications of the TNAA and ELISA assays are (i) to detectthe presence of Ebola Virus RNA and (ii) to detect Ebola IgM and IgG inpatients that are symptomatic or at risk for viral Ebola infection,respectively.

The primary applications of the Electrolyte assays are to accurately andprecisely quantify the concentrations of Sodium and Potassium inpatients presenting with signs and symptoms consistent with infectionwith Ebola virus.

In practicing the methods disclosed herein, whole blood samples arecollected and introduced into a disposable Cartridge at the Field Siteand fed to the SPU, where the samples undergo processing and reactionsteps, and are eventually introduced to a detector to yield a set ofsignals. These signal sets are transferred to the LAS where the raw dataare processed and analyzed and oversight is provided, and the relevantreportables are generated.

In embodiments, Ebola Zaire assays disclosed herein use specific primersto detect genomic sequences for Ebola Zaire Nucleoprotein (EZN) andEbola Zaire Glycoprotein (EZG). For example, the following primers maybe used:

Ebola Zaire Nucleoprotein (TH-EZN) primers: specifically detects genomicsequences encoding Ebola Zaire Nucleoprotein (EZN) in whole bloodspecimens.

Ebola Zaire Glycoprotein (TH-EZG) primers: specifically detects genomicsequences encoding Ebola Zaire Glycoprotein (EZG) in whole bloodspecimens.

Human Centromeric Repeat (TH-HCR) primers: specifically detects humancentromeric repeat (HCR) and is used as a positive control with humanclinical specimens to indicate that adequate isolation of nucleic acidresulted from the extraction of the clinical specimen.

RNA Spike-in Control (TH-RNA-SIC) primers: specifically detectssynthetic template RNA that is spiked into the clinical specimen beforethe RNA extraction process. It is used to indicate that adequateisolation of RNA resulted from the extraction of the clinical specimen.Reagents used in the Ebola Zaire TNAA assay include the following:

In embodiments, Ebola Zaire assays disclosed herein may utilize reagentsfrom the following reagent list:

Volume Reagent Composition Vessel (μL) Lysis Buffer Theranos RNA spikein control RV 120 1000 cp/μL in Chemagen Lysis Buffer Binding Ethanoland other components RV 240 Buffer 2 Wash Buffer 3 Ethanol and othercomponents RV 200 Wash Buffer 4 Ethanol and other components RV 200 WashBuffer 5 Ethanol and other components RV 200 Elution Buffer 10 mMTris-HCl pH 8.0 RV 50 Magnetic M-PVA beads in solution RV 15 BeadsPoly(A) RNA Poly(A) RNA in Poly(A) RNA buffer RV 1.5 Proteinase KDissolved lyophilized proteinase K RV 3.5 TNAA Master T4 buffer, 1.4 mMdNTPs, 0.1% 8X 20 Mix Tween, 400 mM Betaine, Synto Red TNAA 59, 0.8 μMoligonucleotide primers TNAA Enzyme 0.8 U bst, 0.016 RT in T4 buffer RV2 Mix TH EZN-PTC Synthetic RNA in elution buffer TNAA 3 (1000 cp/μL) THEZG-PTC Synthetic RNA in elution buffer TNAA 3 (10,000 cp/μL) T4 Buffer50 mm potassium acetate, 10 mM NA NA magnesium acetate, 1 mM dithio-threitol, 20 mM Tris-HCl, pH 7.9

The TNAA nucleic acid assay uses primers to bind to and detect nucleicacids indicative of Ebola, e.g., indicative of Ebola Zaire strain ofEbola. Primers may be designed to include nucleic acid portions (orportions comprising nucleic acid analogs, peptide nucleic acids, orother molecules that mimic nucleic acids) that are complementary to anysuitable target portion of the Ebola Zaire strain of Ebola. Such primersinclude complementary portions that are typically at least about 8 baseslong, or about 10 bases long, or about 15 bases long, or more.Complementary portions of primers are able to hybridize to targetnucleic acids (e.g., Ebola Zaire strain RNA) and so are useful indetecting the presence of, and measuring amounts of, target nucleic acidin a sample.

In embodiments, control material to be used with the Ebola Zaire assaysdisclosed herein may include the following material.

In embodiments, Ebola TNAA assays disclosed herein include the followingcontrols processed concurrently with every specimen sample:

Ebola Zaire Nucleoprotein Positive Template Control (TH-EZN-PTC): is apositive template control (PTC) designed to react with the TH-EZN primerreagents to indicate whether the Ebola Zaire Nucleoprotein reactionworked. This PTC material consists of synthetic template RNA.

Ebola Zaire Glycoprotein Positive Template Control (TH-EZG-PTC): is apositive template control (PTC) designed to react with the TH-EZG primerreagents to indicate whether the Ebola Zaire Glycoprotein reactionworked. This PTC material consists of synthetic template RNA.

Negative Processing Control (NPC): is a water sample that serves as anegative sample processing control. It is used to ensure no falsepositive reactions for both the TH-EZN assay and the TH-EZG assay in twoseparate reactions.

RNA Spike-in Control (TH-RNA-SIC): is a positive template control (PTC)designed to react with the TH-RNA-SIC reagents to indicate adequateisolation of RNA resulted from the extraction of the clinical specimenand amplification.

Protocol for an Ebola Virus Nucleic Acid Assay

In embodiments, all assay steps for an Ebola virus nucleic acid assaymay be performed on the SPU. For example, sample preparation andextraction processing, amplification, and detection may all be performedon the SPU.

For example, as disclosed herein, magnetic bead-based sample preparationchemistry may be used; in embodiments, all required reagents may beprovided on the assay cartridge, e.g., along with the sample. To avoidsample degradation by RNases in the sample, a sample preservativetechnology may be used, in which the sample is treated with lithiumchloride and iodoacetic acid. The sample preservative reagents arepresent in Thermos Nanotainer™ Tubes (into which the sample may becollected, and which hold the sample immediately after collection) tohelp stabilize the sample upon collection from the patient. Aftercollection, the Nanotainer™ Tube containing a sample may be insertedinto the cartridge, and the cartridge inserted into a SPU, where theliquid handling unit in the SPU processes the sample to extract RNA forthe subsequent pre-amplification reaction followed by the isothermalamplification and detection.

The nucleic acid extraction implemented in the SPU utilizes amagnetic-bead based methodology to isolate and purify the targetedpathogen nucleic acids from the sample. A brief overview of the stepsinvolved in nucleic acid purification as part of the TNAA nucleic acidassay is shown in FIG. 2A. Such steps may be performed by, and may beperformed within, an automatic sample analysis device or system, such asan SPU. The operating temperature of an SPU may be set at, and may becontrolled to be, at all times, close to a set temperature. Inembodiments, the set temperature for operation of an SPU may be, e.g., atemperature selected in the range of from room temperature, or slightlyabove room temperature, to about 40° C., e.g., may be about 26° C., orbe about 27° C., or about 28° C., or about 29° C., or about 30° C., orabout 31° C., or about 32° C., or about 33° C., or about 34° C., orabout 35° C., or about 36° C., or about 37° C., or about 38° C., orabout 39° C., or about 40° C. It will be understood that the temperaturemay exceed such a range for a period of time, or in portions of thesample analysis device or systems, so that, for example, the temperaturemay be about 42° C., or about 44° C., or about 46° C., or about 48° C.,or about 50° C., or about 52° C., or about 54° C., or about 56° C., orabout 58° C., or about 60° C., or about 62° C., or about 64° C., orabout 66° C., or about 68° C., or about 70° C., or higher for a periodof time, or in portions of the device or system within the housing. FIG.2A provides a schematic description of TNAA nucleic acid purificationsteps. In embodiments, such steps are performed in an automatic sampleanalysis device or system (e.g., an SPU) and may be performed in thefield (e.g., in rural or other locations, and not necessarily within alaboratory, clinic, or hospital setting.

The nucleic acid extraction implemented in the SPU utilizes amagnetic-bead based methodology to isolate and purify nucleic acids froma sample matrix.

A brief overview of the steps involved in a magnetic-bead basedmethodology to isolate and purify nucleic acids from a sample matrix isas follows:

1) For blood samples, a Needle tip is inserted into the sample vessel,to access the sample. The sample is mixed by pipetting up and down forseveral cycles.

2) For blood samples, the sample is transferred to the Sonicator vesselby means of two large pipette tips. Lysis buffer (that also inactivatesebola virus in the sample) and functionalized magnetic beads are addedto the Sonicator vessel from other reagent storage locations on theCartridge.

3) Binding buffer, which helps the nucleic acids bind to thefunctionalized magnetic beads, is transferred from a reagent storagewell to the Sonicator vessel and mixed by pipetting up and down.

4) The Magnet Tool that resides inside the SPU is picked up using alarge pipette nozzle. The Magnet Tool is retracted inside of the nozzlesuch that only 2-3 mm is visible before that nozzle is used to pick upthe Magnet Tool Sleeve in the consumable. This sleeve shields the MagnetTool from the sample to prevent contamination.

5) The Magnet Tool is then extended into the tip sleeve and insertedinto the Sonicator vessel to capture the magnet beads on the exterior ofthe sleeve.

6) The magnetic beads with captured nucleic acids aggregate on the tipof the sleeve and can be transported into a well containing wash buffer.

7) The Magnet Tool, covered by the Magnet Tool Sleeve, is retracted intothe nozzle by moving the piston motor, and the nozzle is moved in avertical direction multiple times to release the beads and mix them withthe fluid. The Magnet Tool/Magnet Tool Sleeve is removed from the wellby moving the nozzle.

8) The Magnet Tool is extended back into the tip sleeve and insertedinto the wash buffer well to capture the washed magnet beads andtransport them to the next step.

9) For each additional bead wash to purify the sample, steps 7 through 9are repeated.

10) The Magnet Tool and its sleeve carrying captured magnetic beads withpurified nucleic acid sample is inserted into the elution well.

11) The Magnet Tool is retracted into the nozzle, and the entire nozzleis moved in vertical directions for several cycles to release the beadsand mixed by fluid displacement using a piston motion before retractingthe nozzle such that the tip clears the entire well.

12) The released beads are allowed to incubate in the elution well for 1minute.

13) The Magnet Tool is extended back into the Magnet Tool Sleeve andinserted into the elution buffer well to capture the magnetic beads. TheMagnet Tool Sleeve is then discarded into its original location on theCartridge and the Magnet Tool is returned to its resting location in theSPU.

14) The elution buffer is ready to be distributed into the downstreamTNAA assays.

TNAA Assay and Signal Generation

The elution buffer extracted from the steps above contains the extractednucleic acid material. Detector 3 is brought up to 56° C. utilizing themodule's thermal controller. The TNAA tray with the TNAA vessels ispicked up by the Liquid Handling Module and transferred to Detector 3.The vessels contain the master mix for the TNAA assay, capped with a waxlayer. This wax layer melts at the elevated (56° C.) temperature. 3 uLof elution buffer is aspirated from the elution well on the Cartridgeand transferred into the TNAA vessel using a Mini tip, ensuring that thetip penetrates past the molten wax layer. The sample is mixed with themaster mix to ensure homogeneity. The tip is discarded back into theCartridge. When the tip is moved away from the detector module, thelower temperature of the SPU causes the molten wax around the tip tosolidify, thereby forming a physical barrier around the tip opening andpreventing any sample from leaking out of the tip. This protects againstcontaminating the SPU. A new tip is picked up by the Liquid HandlingModule, and 2 uL of enzyme is transferred from a reagent well in theCartridge to the NAA vessel, and is mixed with the sample and the mastermix. The tip is retracted and returned back to its location on theCartridge.

The reaction mixture in each NAA vessel is incubated for 5 minutes,after which the photodiode corresponding to each reaction vessel is usedto capture the reaction signals of 30 samples sequentially, andcapturing such signals of all 30 samples takes slightly over 10 s. Afterthat there is an approximately 10 s pause before capturing the next set,ensuring that each sample is detected at a frequency of 1 detectionevery 20 s. The data (in the form of counts) is transmitted to the LAS,where the fluorescence signal is recorded and analyzed. The analysisconsists of identifying a change point to determine the inflection timeof the assay.

Controls for TNAA Assay

Controls are included on board the cartridge for the TNAA assay andinclude:

1) No Template Control (NTC): For the TH-EZN assay and TH-EZG assay, aNTC test may be run simultaneously to quality control (QC) forbackground signal and contamination.

2) Positive template controls: for each specimen, positive templatecontrols are run simultaneously for the TH-EZN assay and TH-EZG assaysto ensure general performance of the chemistry and the device.

3) Sample collection and transfer control: Each patient sample willcarry varying amounts of human specific nucleic acid. For each sampleprocessed, a control human centromeric repeat assay is run to verifyappropriate sample collection, sample extraction and device function.

4) Extraction control: A non-natural/synthetic target in the form of RNAis automatically spiked into the sample. This is used as an internalcalibrator to QC sample prep and amplification. This test also checksthe general performance of the chemistry and the device.

ELISA Assays for the Ebola Virus

In embodiments, an assay for Ebola virus may be directed to Anti-ZEBOVIgG, and may include a solid-phase, three step chemiluminescent enzymeimmunoassay. In embodiments, a solid phase, a polystyrene pipette tip,is coated with recombinant EBOV glycoprotein antigen. The patientspecimen is first aspirated by this coated tip. After incubation andwash, an alkaline phosphatase (ALP) conjugated goat anti-human IgG isaspirated by the tip. After incubation and wash, a chemiluminescentsubstrate is aspirated by the tip. This substrate undergoes hydrolysisdue to ALP, the emitted photons are measured by the Luminometer moduleand related to the presence of anti-ZEBOV IgG antibodies in the sample.

In embodiments, Anti-ZEBOV IgM is a solid-phase, four stepchemiluminescent enzyme immunoassay. The solid phase, a polystyrenepipette tip, is coated with mouse anti-human IgM. The patient specimenis first aspirated by this coated tip. After incubation and wash, EBOVglycotprotein antigen is aspirated by this tip. After further incubationand wash, an alkaline phosphatase (ALP) conjugated mouse anti-ZEBOV IgGis aspirated by the tip. After incubation and wash, a chemiluminescentsubstrate is aspirated by the tip. This substrate undergoes hydrolysisdue to, the emitted photons are measured by the Luminometer module andrelated to the presence of anti-ZEBOV IgM antibodies in the sample.

For the ELISA assays, there are positive and negative controls runsimultaneously with the assay each time an assay is run. These controlsare processed in exactly the same way as the patient samples areprocessed (cf. ELISA protocol). The output from these control runs areused to look for any performance deficiencies in the system inreal-time, thereby helping to ensure that the sample output is accurate.If any of the two controls do not pass, the result from that assay maybe considered invalid.

Assay Assay Capture reagent Detection reagent Principle anti-Ebola Mouseanti-human alkaline phosphatase (ALP) ELISA IgM IgM and conjugated mouseanti- recombinant EBOV ZEBOV IgG glycoprotein antigen anti-Ebolarecombinant EBOV alkaline phosphatase (ALP) ELISA IgG glycoproteinantigen conjugated goat anti-human IgG

Reagent List for the Ebola IgG and IgM Assays:

Reagents used in the antibody assays as disclosed herein are describedand listed in the following table (Table 1).

TABLE 1 Ebola IgG and IgM Assay reagent list: VOLUME REAGENT COMPOSITIONVESSEL (μL) Assay diluent buffer 3% BSA in 1x TBS 2 x Wash 100  wellInactivation agent 12% Acetic acid (TBD) RV 50 Neutralization agent 6MNaOH (TBD) RV 50 Assay Wash Buffer 0.1% Tween in 500 mM 24 x RV NaClConjugate: IgG assay Goat anti-human IgG 6 x RV 50 conjugated with ALPConjugate: IgM assay Goat anti-human IgM 6 x RV 50 conjugated with ALPChemiluminescent 12 x RV 50 substrate Positive Control Serum based RV 50Negative Control Serum based RV 50 Coated tips: IgG 12 x Mini NA tip

Protocol for Ebola Antibody ELISAs

The antibody ELISA protocol starts with whole blood samples and extractsplasma for the analytical test. Lysis agents are added to the extractedplasma (and residual cells) to inactivate any ebola virus in the samplebefore further processing in the SPU. The following is the sequence ofsteps for the antibody assays with the extracted, inactivated plasma.

-   -   1. A Large tip is picked up, and 18 uL of diluent is transferred        from the Wash vessel to 4 Round vessels. The tip is ejected back        in the Cartridge.    -   2. 4 Mini tips are picked up, and 2 uL of sample is aspirated in        each tip, and dispensed into the 4 Round vessels.    -   3. The mixture is mixed well by pipetting the fluid back and        forth.    -   4. 4 coated Mini tips, corresponding to the four assays, each        aspirate 10 uL of the mixture, and the mixture is allowed to        incubate with the capture protein on the surface for 10 minutes.    -   5. The mixture is discarded onto an absorbent waste pad located        on the Cartridge, which ensures that the waste fluid is retained        to prevent any possible contamination.    -   6. The 4 tips aspirate 11 uL of wash buffer, followed by a 1        minute incubation, after which the wash buffer is discarded onto        the waste pad.    -   7. Step 6 is repeated 5 more times.    -   8. The 4 tips aspirate 10 uL of detection antibody, followed by        a 5 minute incubation.    -   9. The fluid is discarded onto an absorbent pad, and the wash        steps (steps 7 and 8) are repeated.    -   10. The four reaction tips aspirate 10 uL of ALP substrate from        4 sealed Round vessels containing substrate, followed by a 1        minute incubation.    -   11. The four tips are moved to the Luminometer Module where        luminescence of each of the four tips is detected, and the        corresponding count values transmitted to the LAS.

The count values are analyzed in the LAS, where a calibration functionis applied, analysis is performed on these values and associatedreplicate, control, calibrator, QC, and outlier evaluation, and thefinal antibody results are generated.

Assays for Electrolyte Imbalance Management

Electrolyte Assay Principle:

The electrolyte assays disclosed herein are a subset of generalchemistry assays which may be performed using an automatic sampleanalysis device or system. The general chemistry assays disclosed hereincover common clinical chemistry tests including measuring electrolytes,such as sodium and potassium, renal function tests, liver functiontests, minerals, and metabolites. The sodium and potassium assaysdescribed herein adopt the following methodology: Plasma is separatedfrom whole blood by centrifugation inside the SPU, and the plasma isdiluted in either water or saline, and mixed with reagent(s). Thereaction mixture(s) is incubated as required for each assay. Theproceeding reaction results in a solution which absorbs light at aparticular wavelength, which is detected on Detector 4. This raw signalis transmitted to and analyzed by the LAS to generate the intensityspectra, which is proportional to the concentration of the respectiveanalyte according to Beer-Lambert law.

Sodium Assay—Overview:

Sodium is an essential electrolyte and mineral in the body. It helpskeeps fluids and electrolytes balanced in the body. The concentration ofsodium measured in the blood is a reflection of the amount of sodium andthe amount of water in the vascular space.

The amount of sodium in the body is partially controlled by aldosterone,a hormone made by the adrenal glands. Aldosterone levels in the bodytell the kidneys how much sodium should be excreted and how much sodiumshould be retained in the body. Sodium is also found in a majority offoods and medications. Too much sodium intake may raise a person's bloodpressure and put them at a greater risk for developing heartdisease/heart failure, stroke, and kidney damage. Hyponatremia (lowsodium in the body) is very rare, but most often occurs in people whotake medications which causes increased urination.

Sodium assays are important in assessing acid-base balance, waterbalance, water intoxication, and dehydration. As discussed above,evaluation of sodium levels is important in the evaluation and treatmentof patients presenting with signs and symptoms of Ebola virus infection.

Sodium Assay—Principle of the Method:

In the Sodium colorimetric assay disclosed herein, the sodium dependentenzyme β-galactosidase cleaves the substrateo-nitrophenyl-β-D-galactopyranose, yielding the product onitrophenol, adye with color intensity at 405/420 nm. This absorption is detected bythe SPU, with the raw signal transmitted to the LAS, where it isanalyzed to generate a concentration of sodium based on the rate atwhich o-nitrophenol is formed. This Sodium assay is designed to detectSodium in plasma with a reportable range of about 90-200 mM.

Potassium Assay—Overview:

Potassium is the major cation of the intracellular fluid. It is animportant mineral and electrolyte in the body. It is necessary forproper functioning of the heart, nerves, kidneys, muscles, and thedigestive system. Disturbance of potassium homeostasis (intra- andextracellular) can cause serious health effects. Decreases inextracellular potassium are characterized by muscle weakness,irritability, and eventual paralysis. Cardiac effects includetachycardia, other cardiac conduction abnormalities that are apparent byelectrocardiographic examination, and eventual cardiac arrest.Hypokalemia is common in vomiting, diarrhea, alcoholism, and folic aciddeficiency. Additionally, >90% of hypertensive patients withaldosteronism have hypokalemia. Abnormally high extracellular potassiumlevels produce symptoms of mental confusion; weakness, numbness andtingling of the extremities; weakness of the respiratory muscles;flaccid paralysis of the extremities; slowed heart rate; and eventuallyperipheral vascular collapse and cardiac arrest. Hyperkalemia may beseen in end-stage renal failure, hemolysis, trauma, Addison's disease,metabolic acidosis, acute starvation, dehydration, and with rapidpotassium infusion. As discussed above, evaluation of Potassium levelsis important in the evaluation and management of patients presentingwith signs and symptoms of Ebola virus infection.

Potassium Assay—Principle of the Method:

Potassium is normally maintained in levels from 3.5 to 5.5 mM within thebody and most test methods only read out to 8 mM, making for a verynarrow assay range. Potassium levels are often determined using ionselective electrodes or flame photometry. A potassium assay may use asodium tetraphenylborate method for the determination of potassiumlevels. In this method the compound Sodium Tetraphenylborate reacts withpotassium from plasma. K+ replaces Na+ from Sodium Tetraphenylborate(NaTPB) to form potassium tetraphenylborate, a white precipitate. Theprecipitate remains in suspension with the help of some thickeners andstabilizers, causing an increase in turbidity. The resulting increase inturbidity is detected by the SPU at 450 nm. This raw signal istransmitted to the LAS, where it is analyzed to generate theconcentration of potassium. The assay has a reportable range of about0.1-7.5 mM for Potassium.

Reagent Lists for Sodium Assay and Potassium Assays

Reagents used in the electrolyte assays (sodium assay and potassiumassay) as disclosed herein are described and listed in the followingtable (Table 2).

TABLE 2 Sodium and Potassium Assay reagent list: VOLUME ASSAY REAGENTCOMPOSITION CONCENTRATION (μL) VESSEL Sodium A Cryptand >0.4 mM 1 x 160μL β-D- <8 U/mL 1 Wash Strip galactosidase B O-Nitrophenyl >0.5 mM 3Vessels x 3 vessels β-D- 65 μL galactosidase Medical Decision SerumBased 135 mM 60 Round vessel Level Control 1 Medical Decision SerumBased 150 mM 60 Round vessel Level Control 2 Potassium A Sodium <100 mM1 x 160 μL 1 Wash strip Tetraphenyl Borate Medical Decision Serum Based3.0 mM 60 Round vessel Level Control 1 Medical Decision Serum Based 7.5mM 60 Round vessel Level Control 1

The Ebola assays disclosed herein have been validated using thecomponents referenced above. These Ebola assays were developed usingprimers and probes directed to Ebola Zaire strain viral nucleic acids,and antigens and antibodies indicative of the presence of Ebola Zairestrain virus and of a subject's immune response to such virus.

By way of non-limiting example, FIG. 2B shows a simplified view of oneembodiment of an SPU that may be used herein. The Ebola assays disclosedherein have been validated using automatic sample analysis devices suchas but not limited to those disclosed herein and in, e.g., U.S. Pat. No.8,088,593; U.S. Pat. No. 8,380,541; U.S. Pat. No. 8,435,738; U.S. Pat.No. 8,475,739; U.S. Pat. No. 8,840,838; U.S. patent application Ser. No.13/933,035, filed Jul. 1, 2013; U.S. patent application Ser. No.13/769,820, filed Feb. 18, 2013; U.S. patent application Ser. No.14/183,503, filed Feb. 18, 2014; and U.S. patent application Ser. No.14/214,850, filed Mar. 15, 2014.

Referring still to FIG. 2B, an SPU may be configured to have a display Dwhich may be touchscreen or non-touch screen display. The display D maybe on a top surface, a front surface, an angled top surface, or a sidesurface of the SPU. As seen in FIG. 2B, a cartridge C which may containall reagents, diluents, pipette tips, and/or other disposable used toperform all testing herein, is configured to be received in a cartridgeinsertion location S of the SPU. Optionally, a cartridge C which maycontain substantially all reagents, diluents, pipette tips, and/or otherdisposable used to perform all testing herein, is configured to bereceived in a cartridge insertion location S of the SPU. In one examplethe cartridge C may contain all reagents except perhaps a common diluent(such as water or the like) or a common reagent used in processing.Optionally, at least some pipette tips in the cartridge C are coated(inside of tip or outside of tip) with an ebola antibody such asdescribed herein. In one non-limiting example, the cartridge C isconfigured with assay(s) for diagnostic marker(s) and assay(s) forprognostic marker(s) for the disease at issue.

Optionally, some embodiments may use a further containment enclosure Eabout the SPU which in manner similar to a fume hood glove box, mayinclude one or more ports or other devices that allow for protectedaccess. In one non-limiting example, the enclosure E may be configuredto have hardware to implement a negative pressure environment tominimize the risk of contaminants escaping the enclosure. Optionally,some embodiments may configure the SPU to have the negative pressureenvironment within the SPU. In embodiments, a containment enclosure Emay include one or more air filters, e.g., high efficiency particulateair (HEPA) filters suitable for blocking the flow of contaminants andmicro-organisms. In embodiments, such air filters may filter outside airas it flows into, or may filter internal air as it leaves, or both, theinterior of the containment enclosure. Such filters may be effective toreduce or prevent the entry of dust or other contamination into the SPU,and to reduce or prevent contamination from exiting the SPU (includingreducing or preventing contamination from samples from exiting anenclosure E).

The assays have been completed within 1 hour of loading the sample intothe cartridge and inserting the cartridge into the automatic sampleanalysis device.

Assay Results

The results of TNAA assays directed at Ebola virus (Ebola Zaire strain)are shown in FIGS. 3-8. The data shown in FIG. 3 demonstrate performancedata of the Ebola Zaire TNAA Assay. These data are for the TH-EZNreaction which detects the presence of the Ebola Zaire nucleoproteinRNA. FIG. 3 shows results from titration experiments using synthetic RNAtarget for Ebola Zaire nucleoprotein. The y-axis shows the time ofdetection based on the fluorescent signal recorded in real-time duringthe TNAA amplification reaction. The time-cutoff for assessing apositive reaction was 45.6 minutes. As few as 10 copies/uL (cp/uL) ofsynthetic Ebola Zaire nucleoprotein RNA was detectable in theseexperiments.

FIG. 3 plots the detection time in minutes (vertical axis) versus thecopies per microliter (cp/uL) for four conditions: 1000 copies permicroliter (cp/μL) of Ebola Zaire nucleoprotein RNA (left-most column),100 cp/μL of Ebola Zaire nucleoprotein RNA (second from left column), 10cp/μL of Ebola Zaire nucleoprotein RNA (second from right column), and“NTC” (no template control) in which no Ebola Zaire nucleoprotein RNAwas present (right-most column). As shown in FIG. 3, the presence ofEbola RNA was detectable in less than 30 minutes for samples having 1000cp/μL of Ebola Zaire nucleoprotein RNA; Ebola RNA was detectable inslightly more than 30 minutes for samples having 100 cp/μL of EbolaZaire nucleoprotein RNA; even in samples having only 10 cp/μL of EbolaZaire nucleoprotein RNA, Ebola RNA was detectable in less than 40minutes. For comparison, (non-specific) signals from samples with noEbola Zaire nucleoprotein RNA were not detected for times greater thanabout 70 minutes, and so are clearly distinguishable from clinicallysignificant signals indicative of the presence of Ebola RNA.

The data shown in FIG. 4 demonstrate the performance of the Ebola Zairenucleoprotein TNAA Assay in the presence of possibly cross-reactingsubstances. No false positive reaction was observed, as the detectiontime for all possibly cross-reacting pathogens was substantially longerthan the cutoff time (45.6 minutes). The detection time for Ebola ZaireRNA at 100 cp/μL was substantially below the cutoff time, indicatingpositive detection of the target viral nucleic acid in the sample. Nocross reactivity of the TH-EZN reaction to other pathogens in the assayshown was observed.

In FIG. 4, as in FIG. 3, the y-axis shows the time of detection based onthe fluorescent signal recorded in real-time during the TNAAamplification reaction. Thus, the vertical axis plots detection time (inminutes) based on the fluorescent signal recorded in real-time duringthe TNAA amplification reaction. Columns heights indicate the time inminutes until an inflection in the measured fluorescent signal wasidentified; each column is labeled with the name of the pathogen nucleicacid presented for detection. The column labeled “Ebola Zaire 100 cp/uL”is the positive control. The non-template control is labeled “NTC” (noRNA target added). Natural or synthetic pathogen nucleic acids testedincluded nucleic acids from several forms of human immunodeficiencyvirus (HIV), Herpes Simplex Virus 1 (HSV-1), adenovirus SC,cytomegalovirus (CMV), Epstein-Barr virus (EBV), candida albicans, humanpapilloma virus (HPV), herpes simplex virus (HSV), hepatitis B virus(HBV), and other pathogens. The cutoff for assessing a positive reactionwas 45.6 minutes; only the signal for detection of Ebola Zaire was belowthe cutoff, indicating a positive reaction. It is notable that, for allpathogen RNA targets except Ebola Zaire, the column heights are similarto that of the NTC column height, indicating no detectablecross-reactivity.

As shown in FIG. 5, the specificity of the TH-EZN reaction was tested byadding the listed pathogens to the sample in addition to the positivecontrol synthetic templates. No false negative reactions were seen. Thepositive control is labeled “Ebola Zaire 100/cp/ul”. The non-templatecontrol is labeled “NTC”. The y-axis shows the time of detection basedon the fluorescent signal recorded in real time during the TNAAamplification reaction. The cutoff for assessing a positive reaction was45.6 minutes.

The data shown in FIG. 5 demonstrate the specificity of the Ebola Zairenucleoprotein TNAA Assay in the presence of possibly interferingsubstances. The specificity of the TH-EZN assay is quite good, as thedetection time is not much affected by the presence of other pathogens,as shown in the figure. The same possible interfering pathogens astested in the experiments shown in FIG. 4 were used in the experimentsshown in FIG. 5 (forms of human immunodeficiency virus (HIV), HerpesSimplex Virus 1 (HSV-1), adenovirus SC, cytomegalovirus (CMV),Epstein-Barr virus (EBV), candida albicans, human papilloma virus (HPV),herpes simplex virus (HSV), hepatitis B virus (HBV)).

The Table shown in FIG. 6 further illustrates the experiments performedwhich demonstrate the specificity of the Ebola Zaire nucleoprotein TNAAAssay in the presence of possibly interfering substances. The effect ofinterfering substances was tested for the TH-EZN reaction in twomanners. First interfering substances were added to the sample inaddition to the positive control synthetic templates to see if thesubstances interfered with the reaction. Second, interfering substanceswere added to a sample without any template (NTC) to assess if anon-specific reaction occurred. Each substance was tested at two levels(lx concentration and 0.1× concentration). As shown in FIG. 6, none ofthe possibly interfering substances prevented the detection of syntheticEbola Zaire nucleic acid (columns labeled “inhibits true positive”). Asshown in FIG. 6, only one of the possibly interfering substances, andonly at the higher concentration, caused a false positive (hemoglobin at5 g/L concentration). Thus, the Ebola Zaire TNAA Assay is quite robust.

The Ebola Zaire ELISA assays for detecting the presence of anti-Ebolavirus IgG and IgM antibodies in samples (ZEBOV IgG and ZEBOV IgM assays,where ZEBOV stands for Zaire Ebola Virus), along with the Ebola ZaireELISA assays for detecting the presence of GP antigen in samples (ZEBOVGP antigen assay), are suitable for the qualitative detection of IgM andIgG antibodies for Ebola virus and the quantitative measurement of ZEBOVGP antigen, all from EDTA anti-coagulated plasma (extracted from wholeblood specimens). EDTA coagulated whole blood or plasma may be obtained,for example, by fingerstick, or may be obtained by other methods ofdrawing blood samples. These assays may also be performed on serumsamples, which may be obtained, for example, by venipuncture. All ofsuch samples may be obtained, for example, from individuals in affectedareas with signs and symptoms of Ebola virus infection.

FIGS. 7A and 8A show the results of Ebola Zaire ELISA assays. The datashown provide performance data of the Ebola Zaire ELISA IgG and IgMAssays, and demonstrate detection of targets in the samples. FIG. 7Aprovides a comparison of in-house (negative) EDTA-anticoagulated plasmasamples and high-concentration spiked samples. All samples in FIG. 7Awere spiked with human anti-ZEBOV IgG to a nominal concentration of 1ug/ml. FIG. 8A provides a comparison of in-house (negative)EDTA-anticoagulated plasma samples and low-concentration spiked samples.All samples in FIG. 8A were spiked with human anti-ZEBOV IgM to anominal concentration of 0.3 ug/ml. As discussed above, the IgG and IgMassays (ELISA assays) may be performed in heparin-anticoagulated plasmasamples or in EDTA-anticoagulated blood samples (e.g., whole blood orplasma); such samples may be collected by a fingerstick as whole blood,and then EDTA or heparin applied to whole blood or plasma.

For the qualitative assays (Ebola Zaire ELISA IgG and IgM Assays), theassay output is antibody index or antibody titer, which is directlyrelated (but may not be quantitatively identical to) antibody titer. Theantibody index is a continuum as opposed to the final output of theassay which is discrete/categorical. For the ZEBOV GP antigen assaywhich is a quantitative assay the measuring interval will span 2500-25ng/ml of the GP antigen.

Anti-ZEBOV IgG assay: FIG. 7B shows a dose response as demonstrated byspiking pooled normal serum samples with a human anti-ZEBOV IgG clone atconcentrations ranging from 0.05-500 ug/mL. EDTA plasma samples fromn=40 in house normal donors was also tested. FIG. 7B compares assayresults from negative and spiked samples; the figure shows signals fromassays run with in-house (negative) EDTA-anticoagulated plasma samples(squares) and spiked samples. All samples were spiked with humananti-ZEBOV IgG. Putative cut-off set at signal 1.8 corresponds to asensitivity of 0.5 ug/ml. The antigen used in this anti-ZEBOV IgG assayshows reactivity for antibodies against glycoprotein as well as otherproteins of ZEBOV. Mild cross reactivity with Sudan EBOV IgG is alsoseen. Cross reactivity data is summarized in the Table shown in FIG. 7C.

As discussed above, FIG. 8A compares ZEBOV IgM ELISA assays results from(negative) EDTA-anticoagulated plasma samples with results fromlow-concentration spiked samples. FIG. 8B also compares assay resultsfrom negative and spiked samples, showing results from the Anti-ZEBOVIgM assay; the figure shows signals from assays run with in-house(negative) EDTA-anticoagulated plasma samples (squares) and spikedsamples. The dose response shown in FIG. 8B is demonstrated by spikingpooled normal serum samples with a human anti-ZEBOV IgM control atconcentrations ranging from 1 to 10 μg/mL. EDTA plasma samples from 12normal donors was also tested. Putative cut-off was set at signal 1.5.All samples were spiked with human anti-ZEBOV IgM.

FIG. 9A shows dose-response results from assays with recombinant ZEBOVGP antigen spiked pooled normal serum calibrators. Recombinant ZEBOV GPantigen was spiked into pooled normal donor serum at concentrationsranging from 2.5 μg/mL to 25 ng/mL. FIG. 9A shows the dose responsecurve of the assay. The antibody pair used in this assay is veryspecific for the ZEBOV GP antigen and does not cross-react with the GPantigen from the Marburg genus virus. There is also no cross-reactivityseen with GP antigens form the other species of Ebola virus, and againstother viral proteins in the ZEBOV genome. This cross-reactivity data issummarized in the Table shown in FIG. 9B.

Methods of Interpreting the Assay Results

All test controls should be examined prior to interpretation of patientresults. If the controls are not valid, the patient results cannot bereliably interpreted. Assessment of clinical specimen test resultsshould be performed after the positive and negative controls have beenexamined and determined to be valid and acceptable. Assay results may becompared to Ebola Zaire TNAA assay positive and negative controls, andmay be reported as positive, equivocal, or negative for Ebola Zaire.

Interpretation of Nucleic Acid Assay Results:

Both TH-EZN and TH-EZG reactions should be positive for a positive Ebolatest result. If only one of these reactions is positive, then theresults are inconclusive and a retest is required. Both TH-EZN andTH-EZG reactions should be negative for a negative Ebola test result.

In order for a test run to be valid, all NTC reactions must be negative.If one or more NTC fails, the entire run is invalid and potentialsources of contamination should be identified and corrected, and thespecimen retested. Failure to achieve a positive PTC reaction for any ofthe assays (TH-EZN-PTC and TH-EZG-PTC) invalidates the run.

The TH-RNA-SIC assay and TH-HCR assay should both be positive. All runsshould exhibit a positive TH-RNA-SIC assay. A negative TH-RNA-SIC assaycould indicate improper RNA extraction, low recovery of RNA, improperreaction, reagent or equipment malfunction, or amplification inhibition.

All clinical specimens should exhibit a positive HCR reaction,indicating sufficient recovery of acceptable quality RNA from thespecimen. A negative TH-HCR reaction could indicate improper RNAextraction, low recovery of RNA, improper reaction, reagent or equipmentmalfunction, amplification inhibition, or absence of sufficient humancellular material in the specimen to enable detection.

In embodiments, positive results for Ebola Zaire assays disclosed hereinshould override a failed (negative or uncertain) TH-RNA-SIC assay orTH-HCR assay result for that clinical specimen. In embodiments, a failedTH-RNA-SIC assay or TH-HCR assay reaction that accompanies a negative oruncertain target result requires retesting.

The following table provides an overview of nucleic acid assay resultsand their interpretation.

TH- TH- TH- TH- TH- Inter- Fol- TH- TH- EZN- EZG- EZN- EZG- RNA- TH-pre- low- EZN EZG PTC PTC NTC NTC SIC HCR tation up + + + + − − + +Positive − − + + − − + + Neg- ative + − + + − − + + Incon- Retestclusive − + + + − − + + Incon- Retest clusive − Invalid Retest Run −Invalid Retest Run + Invalid Retest Run; possible contam- ination +Invalid Retest Run; possible contam- ination − − − Extrac- Retest tionor Reagent Failure − − − Invalid Retest run or low samplequality + + + + − − + − Positive + + + + − − − + Positive + + + + − − −− Positive

Interpretation of Antibody Assay Results:

Interpretation of the results of assays for anti-Ebola antibodies (ELISAassays) are discussed in the following.

A negative Anti-ZEBOV IgG result indicates no evidence of recent of pastEbola infection. Results must be interpreted in the context of clinicalpresentation. A negative Anti-ZEBOV IgG result does not rule out otheretiologies.

A positive Anti-ZEBOV IgG result indicates recent or chronic Ebolainfection. Results must be interpreted in the context of clinicalpresentation. A positive Anti-ZEBOV IgG result does not rule outco-infection or other etiologies.

An equivocal Anti-ZEBOV IgG result cannot determine Ebola status.Retesting of the subject after 1-2 weeks is indicated if symptomspersist.

A negative Anti-ZEBOV IgM result indicates no evidence of acute Ebolainfection. Results must be interpreted in the context of clinicalpresentation. A negative Anti-ZEBOV IgM result does not rule out otheretiologies.

A positive Anti-ZEBOV IgM result indicates acute Ebola infection.Results must be interpreted in the context of clinical presentation. Apositive Anti-ZEBOV IgM result does not rule out co-infection or otheretiologies.

An equivocal Anti-ZEBOV IgM result may not determine Ebola status.Retesting of the subject after 1-2 weeks is indicated if symptomspersist.

Controls: Each assay (Ebola IgG and Ebola IgM) is run with a positiveand a negative control. If any of the controls do not pass, the resultsfrom that run may be considered invalid.

A ZEBOV GP antigen assay result that is greater than the lower limit ofquantification (LLOQ) of the assay indicates an acute Ebola infection.Results must be interpreted in the context of clinical presentation,e.g., indication of an acute Ebola infection does not rule outco-infection or other etiologies as well.

A ZEBOV GP antigen assay result that is lower than the lower limit ofquantification (LLOQ) of the assay indicates no evidence of an acuteEbola infection. Results must be interpreted in the context of clinicalpresentation, e.g., such a result does not rule out other etiologies.

Controls: Each ZEBOV GP antigen assay may be run with a positive and anegative control. If any of the controls do not pass, the results fromthat run may be considered invalid.

Interpretation of Electrolyte Assay Results:

Interpretation of the results of assays for sodium and for potassium arediscussed in the following.

Sodium and potassium results should be interpreted based on routineclinical practice and help guide the care provider in administeringsupportive care including oral and/or intravenous (IV) fluids tomaintain fluids and electrolytes.

Controls: Each assay (sodium and potassium) is run with a low and a highcontrol. If any of the controls do not pass, the results from that runshould be considered invalid.

Measurement of sodium levels in samples obtained from subjects suspectedof suffering from an infectious disease such as Ebola, e.g., Ebola Zairestrain, is useful in providing and managing the treatment of thatsubject. Measurement of potassium levels in samples obtained fromsubjects suspected of suffering from an infectious disease such asEbola, e.g., Ebola Zaire strain, is useful in providing and managing thetreatment of that subject. Measurement of electrolytes such as sodiumand potassium may be of particular use in providing and managing thetreatment of subjects suffering from hemorrhagic diseases, such asEbola, or other diseases which cause fluid loss (e.g., cholera).

Example 2

Nucleic acid sequences of Ebola viruses include, for example, EbolaZaire nucleoprotein nucleic acid sequence, NCBI GenBank AF272001 EbolaZaire (Mayinga strain) Nucleoprotein (SEQ ID NO: 1). (The nucleic acidsequence of SEQ ID NO: 1, and the other nucleic acid sequences providedherein, are written as the DNA sequence (i.e., where the RNA has uracil(u) the DNA has thymine (t)). Nucleic acid sequences encoding Ebolavirus proteins include, for example, Ebola Zaire nucleoprotein (NCBIGenBank J04337.1 Ebola Zaire (Mayinga strain) (SEQ ID NO: 2)), andinclude, for example, the nucleic acid sequence encoding Ebola virusglycoprotein (NCBI GenBank U23187.1 Ebola Zaire (Mayinga strain) (SEQ IDNO: 3).

The amino acid sequences of Ebola virus proteins include thenucleoprotein amino acid sequence encoded by the above Ebola Zairenucleoprotein, which has the amino acid sequence found in NCBI GenBankJ04337.1 Ebola Zaire (Mayinga strain) (SEQ ID NO: 4). A further Ebolavirus nucleoprotein amino acid sequence, for example, is Ebola Zairenucleoprotein amino acid sequence, Uniprot Accession No. P18272 (versionP18272.2, GI 6136281) (SEQ ID NO: 5). The amino acid sequences of Ebolavirus proteins include, for example, the glycoprotein sequence encodedby the nucleic acid sequence mentioned above, which has the amino acidsequence found in NCBI GenBank U23187.1 Ebola Zaire (Mayinga strain)(SEQ ID NO: 6).

Further Ebola virus nucleic acid sequences (from which the amino acidsequences of Ebola virus proteins may be determined) are found, forexample, listed by the National Center for Biotechnology Information(NCBI) (main URL: http://www.ncbi.nlm.nih.gov/) Bioproject for Zaireebolavirus (Zaire ebolavirus genome sequencing) Accession No.PRJNA257197, ID No. 257197, all of which sequences are herebyincorporated by reference in their entireties. The amino acid sequenceslisted therein, the amino acids encoded by these nucleic acid sequences,and portions thereof, including C-terminal portions, may be antigenic,leading to anti-Ebola antibodies when present in an infected individual,and when presented to an experimental animal or organism in anartificial (e.g., laboratory-based) method for producing antibodies.Antibodies to Ebola virus antigens are discussed, for example, in VirusResearch 176(0):83-90 (2013), which is hereby incorporated by referencein its entirety.

While the assays, methods, kits, devices, and systems have beendescribed and illustrated with reference to certain particularembodiments thereof, those skilled in the art will appreciate thatvarious adaptations, changes, modifications, substitutions, deletions,or additions of procedures and protocols may be made without departingfrom the spirit and scope of the invention. By way of example and notlimitation, although many embodiments are described in the context ofusing TNAA, it should be understood that some other embodiments ofsystems, methods, devices, and/or kits herein may use other types ofnucleic acid amplification (isothermal and/or thermal-cycled) currentlyknown or other types of nucleic acid detection methods that may bedeveloped in the future. It should also be understood that although manyof the embodiments herein are described in regards to Zaire ebolavirus,the embodiments herein can be applied to other strains currently known(Sudan, Reston, Tai Forest, or Bundibugyo) or may be discovered in thefuture. It should also be understood that many of the samples analyzedherein are blood samples (capillary, venous, arterial, etc. . . . ),some embodiments may also be configured to process samples from oralswabs or tissues samples (spleen, liver, skin snips) that may beobtained by techniques such as biopsy. It should also be understood thatembodiments herein may involve analysis of samples with EDTA and/orsamples with heparin (from same subject). Samples to be transported forprocessing may be stored at about 4° C., optionally in the range ofabout 2° C. to about 6° C.

Example 3 Analytical Sensitivity of the ZEBOV GP Antigen Assay

The Zaire Ebola virus (ZEBOV) GP antigen assay is an antigen captureELISA that is highly specific for the Zaire Ebola virus glycoprotein.The assay uses low sample volume and is designed for automated analysisof the ZEBOV GP antigen detection. The reagent coated on the surface ofthe Zaire Ebola Virus (ZEBOV) GP antigen assay is a protein A-purifiedmouse monoclonal antibody (clone 4F3) reactive to EBOV GP. The antibodydetects GP in virus-like particles (VLP) and recombinant GP without thetransmembrane region (rGPdTM). It is a monoclonal antibody of the IgG2aisotype. This antibody shows no cross-reactivity to Sudan virus (SUDV)or Marburg Virus (MARV) VLP or MARV rGPdTM. The antibody that is used asthe detection antibody in the Zaire Ebola Virus (ZEBOV) GP antigen assayis a protein A-purified neutralizing human monoclonal antibody (KZ52)derived from a human convalescent patient who survived an EBOVinfection. KZ52 is directed towards EBOV GP. The antibody detectsrecombinant EBOV GP without the transmembrane region (EBOV rGPdTM)expressed in both mammalian and insect cells. It is a human variable,human constant of the IgG1 isotype. This antibody shows no crossreactivity against Marburg virus (MARV) GP. The Zaire Ebola Virus(ZEBOV) GP antigen assay is very specific to the envelope bound GP andrecombinant insect cell expressed GP with the truncated transmembranedomain and shows no cross reactivity against the soluble GP form whichis released into the medium of EBOV-infected cells. This is because KZ52antibody that is used as detection antibody in the assay bindsexclusively to full length GP since the epitope is known to compriseamino acids present in both GP1 and GP2 subunits.

Limit of Blank (LoB):

The cut-off was established as the RLU value at the limit of the blank.Capillary samples were collected in Theranos Nanotainer™ Tubes fromin-house subjects. Nanotainer™ Tubes were loaded on ZEBOV GP antigen,field locations cartridges and processed directly on SPU. RLU valueswere measured from 25 different ZEBOV GP negative capillary samplesanalyzed on the ZEBOV GP antigen assay. The maximum RLU value from thisdata set was 9.93. This value was rounded up to set the cut-off at 10RLU. In other words, any sample greater than 10 RLU, would yield aCut-off index (COI) of greater than 1.0, and would be classified asequivocal or positive. The equivocal zone and the rationale behind it isconsidered below.

Limit of Detection (LoD):

Inactivated viral isolate obtained from the CDC was used fordetermination of LoD. This gamma-irradiated viral stock is prepared fromviral supernatant by the CDC from the Zaire Ebolavirus, Mayinga 1976strain. This inactivated viral stock was reported at 2.5×10⁸ TCID₅₀/ml.

K2-EDTA anti-coagulated venous whole blood was aliquoted into TheranosNanotainer™ tubes. Different concentrations of titered viral stock werespiked into the Nanotainer™ Tube. The Nanotainer™ Tubes were loaded onZEBOV GP antigen, field locations cartridges and processed directly onSPU. The results are tabulated in Table N-2.

Table N-2 provides a summary of the response of ZEBOV GP antigen assayto serial dilutions of inactivated viral isolate (from CDC).

TABLE N-2 No. of Virus Titer positives Virus Strain (10⁷ TCID₅₀/ml)called % Positive ZEBOV, Mayinga 1976 25 3 100 ZEBOV, Mayinga 1976 2.5 3100 ZEBOV, Mayinga 1976 1.25 3 100 ZEBOV, Mayinga 1976 0.83 3 100 ZEBOV,Mayinga 1976 0.625 3 100 ZEBOV, Mayinga 1976 0.500 2 66 ZEBOV, Mayinga1976 0.417 1 33

The lowest COI for lowest virus titer was 1.5, very close to 1. Based onthese data, the tentative LoD is 6.25×10⁶ TCID₅₀/ml.

In order to compare this LoD to LoD of a nucleic acid amplificationassay and other ELISA assays found in literature, Towner et al (Journalof Virology, April 2004, p. 4330-4341) may be considered. Towner et al.studied sequential samples obtained from symptomatic patients duringEbola outbreak in Uganda in 2000-2001. In the Towner et al. study, theauthors analyzed a subset of serum samples using a plaque assay(PFU/ml), a Q-RT-PCR assay (copies/ml) and an ELISA (OD_(sum)).Comparing the RNA copy number and OD_(sum) (transformed into COI for theauthors' assay) measured on the same samples, the following correlationwas obtained (See Table N-3):

COI_(ELISA)=0.0012(RNA copies/ml)^(0.44)

This correlation implies that the LoD of the ELISA assay (COI_(ELISA)=1)corresponds to 4.45×10⁶ copies RNA/ml. Assuming 1 copy RNA/ml=1TCID₅₀/ml for this virus, we estimate the LoD of the ELISA assay ofTowner et al to be 4.45×10⁶ TCID₅₀/ml. This number is of the same orderas the tentative LoD for the present ZEBOV GP antigen assay determinedabove using gamma-irradiated inactivated Ebola virus: 1 COI=6.25×10⁶copies RNA/ml, and 1 COI≡2.1×10³ PFU/ml. Table N-3 presents a summary ofmeasurements on EBOV clinical samples by different methods from Towneret al. The cut-off of this ELISA assay was set at OD_(sum)=0.45(http://www.ncbi.nlm.nih.gov/pmc/articles/PMC374287/pdf/1940.pdf).

TABLE N-3 Sample ID PFU RNA log10(RNA) OD_(sum) COI 1469 5.00E+012.00E+05 5.3 0.11 0.24 1522 1.50E+04 1.80E+07 7.3 0.82 1.82 15274.30E+05 4.00E+08 8.6 3.54 7.87 1591 1.60E+06 3.30E+09 9.5 7.38 16.41612 5.30E+05 2.50E+08 8.4 7.46 16.60

Confirmation of LoD:

LoD of 6.25×10⁶ TCID₅₀/ml was confirmed by analyzing 20 more replicates.Capillary samples were collected in Nanotainer™ Tubes from in-housesubjects. These samples were spiked with viral isolate to nominally havevirus concentration at the LoD. The Nanotainer™ Tubes were loaded onZEBOV GP antigen, field locations cartridges and processed directly on aSPU. The results obtained from 25 ZEBOV GP negative samples, 6 samplesbetween blank and the LoD, and 23 samples spiked at the LoD wereanalyzed on the ZEBOV GP antigen assay. Twenty two (22) samples testedpositive, i.e. above the cut-off. One sample was in the equivocal zone(discussed below). The LoD of 6.25×10⁶ TCID₅₀/ml is thus validated.

Equivocal Zone:

In the zone between the upper limit of the blank (cut-off) and the lowerlimit of the LoD replicates, the signal is too high to be a true blank,but too low to be called a certain positive. This zone was designated asthe equivocal zone. Samples with antigen levels between the blank andthe LoD will lie mostly in this zone.

Example 4 Analytical Specificity of the ZEBOV GP Antigen Assay

Reactivity:

Reactivity of the ZEBOV GP antigen assay was evaluated for additionalisolates/recombinant antigens of Ebola virus and related families.

As seen in Table N-4, the ZEBOV GP antigen assay does not showreactivity to soluble sGP antigen. The Zaire Ebola Virus (ZEBOV) GPantigen assay is highly specific to the envelope bound GP and therecombinant insect cell expressed GP with the truncated transmembranedomain. This is because KZ52 antibody that is used as detection antibodyin the assay binds exclusively to full length GP and the epitope isknown to comprise amino acids present in both GP1 and GP2 subunits. Thisepitope does not exist in sGP. Table N-4 presents reactivity informationfor the ZEBOV GP antigen assay as disclosed herein.

TABLE N-4 ZEBOV GP antigen assay results Detected/Not Strain TestedConcentration Detected ZEBOV culture cell slurry, Not determined. Cell3/0 Mayinga 1976 strain, gamma- slurry was used neat irradiated,provided by CDC ZEBOV, Mayinga 1976 strain. 2.5 × 10⁸ TCID₅₀/ml 3/0 Thissame material was used for LoD sGP, recombinant, spiked 2.5 ug/ml 0/3Reston EBOV GP, recombinant, 25 ug/ml 0/1 spiked Sudan EBOV GP,recombinant, 25 ug/ml 0/1 spiked ZEBOV VP40, recombinant, 25 ug/ml 0/1spiked Angola MARV GP, 25 ug/ml 0/1 recombinant, spiked Bundibugyo EBOVGP, 25 ug/ml 0/1 recombinant, spiked Musoke MARV GP, 25 ug/ml 0/1recombinant, spiked ZEBOV NP, recombinant, 25 ug/ml 0/1 spiked

Cross Reactivity:

In order to identify microorganisms that may be expected to be presentin blood samples from West Africa travelers, a literature review wasundertaken. Infectious Diseases: A Geographic Guide, Editor(s): EskildPetersen, Lin H. Chen, Patricia Schlagenhauf, Wiley was identified as auseful source of information. From this review, a summary of infectiousdiseases commonly observed in West Africa was compiled; this summaryincludes the following.

Travelers from West Africa (including Western tourists/businessmen,Africans resident in industrialized countries who return to theircountry of origin to visit friends and relatives and migrants of Africanorigin) typically present with three conditions: fever,gastroenterological disorders and dermatological problems.Microorganisms observed to be responsible for these conditions arediscussed in the following.

Fever: About 3-11% of travelers report occurrence of fever, with up to50% presenting within first week of return and 96% within 6 months.Fevers are further classified as undifferentiated and differentiatedfevers. Undifferentiated fevers include Malaria, P. falciparum, and morerarely P. ovale, or P. vivax. Also rare are Viral hemorrhagic fever(VHF), which may be caused by Marburg, Ebola and Lassa viruses.Differentiated fevers include Fevers with Rash, such as Measles,Rubella, Neisseria meningitidis, Rickettsia (rare), Dengue, West Nile,Chikungunya (occasional), Cytomegalovirus, Toxoplasma, Schistosomiasis,HIV, and Treponema Pallidum. Fevers with Respiratory symptoms includeStreptococcus Pneumoniae, Haemophilius influenzae, group Astreptococuus, influenza virus, Tuberculosis, Ascaris, Strongyloides,hookworm. Fever with Gastrointestinal symptoms include enteric fever.Fevers with Jaundice include Acute hepatitis (A, B, E), leptospirosis,Shigella. Fevers with Neurological symptoms include Neisseriameningitides, S. pneumonia, H influenzae type B, enterovirus,herpesviruses. Gastrointestinal disorders include E. Coli, Salmonellaspp., Shigella spp., Campylobacter, rotavirus and noroviruses, Giardialamblia, Cryptosporidium spp. Skin conditions include filariasis, guineaworm, mansonellosis, Mycobacterium ulcerans.

These conditions were further reviewed to evaluate the probability ofobserving the causative microorganisms in blood samples duringinfection. The following organisms were identified as unlikelycandidates for appearing in a subject's bloodstream: Measles, Rubella,Schistosomiasis, Treponema Pallidum, Influenza Virus, Hookworm,Leptospirosis, S. pneumonia, H. influenzae type B, Enterovirus, E. coli,Salmonella, Campylobacter, Rotavirus, Norovirus, Giardia lamblia,Filariasis, Mansonellosis.

Other cross-reactants are more likely to be present in subject'sbloodstream. Within the constraints of availability from commercialsources and lack of ability to culture some of the BSL3/3+/4micro-organisms, the organisms/proteins listed in Table N-5 wereselected for testing.

For cross-reactivity testing, either in-house cultures or commercialcultures with known titers were used. All viruses were spiked atconcentrations greater than 10⁶ TCID₅₀/ml or at the maximum possibleconcentration allowed by the available stock. All bacteria were spikedat concentrations greater than 10₆ CFU/ml or at the maximum possibleconcentration allowed by the available stock. After spiking, Nanotainer™Tubes were loaded on ZEBOV GP antigen, field locations cartridges andprocessed directly on a SPU. Results are summarized in Table N-5 below,which provides a summary of cross-reactivity studies on ZEBOV GP antigenassay.

TABLE N-5 Results Virus/Bacteria/Parasite Strain Concentration Units(Detected X/3) Human Rotavirus Wa 5 × 10⁶ TCID₅₀/ml 0/3 AdenovirusSerotype 3 2 × 10⁷ TCID₅₀/ml 0/3 Enterovirus 68 Fermon 1 × 10⁵ TCID₅₀/ml0/3 Pseudomonas aeruginosa Z139 1 × 10⁷ CFU/ml 0/3 Streptococcuspneumoniae Z022 1 × 10⁷ CFU/ml 0/3 Hemophilus influenzae MinnA 1 × 10⁷CFU/ml 0/3 Neisseria meningitidis Serogroup A 1 × 10⁷ CFU/ml 0/3 LassaVirus, NP Protein 100 ug/ml 0/3 Recombinant Rift Valley fever virus,Glycoprotein 100 ug/ml 0/3 Recombinant Influenza A H3N2 A/Texas/50/20121 × 10^(5.15), neat TCID₅₀/ml 0/3 Influenza B B/Wisconsin/1/2010 1 ×10^(5.23), neat TCID₅₀/ml 0/3 Salmonella enterica Z005 1.19 × 10⁹  CFU/ml 0/3 Typhimurium Vibrio cholera Z132 1.8 × 10⁹   CFU/ml 0/3Yersinia enterocolitica Z036 1.16 × 10⁹   CFU/ml 0/3 Shigella boydiiZ131 4.63 × 10⁸   CFU/ml 0/3 Plasmodium vivax South Vietnam Unknown, 0/3neat Dengue Virus Type 4 H241   1 × 10^(6.77) TCID₅₀/ml 0/3 Yellow Fevervirus 17D 1 × 10^(5.86), neat TCID₅₀/ml 0/3 (Heat Inactivated)Chikungunya (Heat R80422 1 × 10^(5.86), neat TCID₅₀/ml 0/3 Inactivated)Plasmodium Falciparum Source: E. Coli 100 ug/ml 0/3 Cs Mosaic protein,Recombinant Plasmodium Falciparum Source: E. Coli 100 ug/ml 0/3 HSP70protein, Recombinant Crimean Congo NP Protein  50 ug/ml 0/3 HemorrhagicFever virus, Recombinant

Interfering Substances:

The impact of potentially interfering substances on the ZEBOV GP antigenassay was evaluated. The evaluation was conducted to demonstrate thatthe potential interferents do not generate false positive results inknown negative specimens, and do not lead to false negative results inknown positive specimens.

K2-EDTA anti-coagulated venous samples were collected from in-housesubjects. These samples were spiked with interferents at theconcentrations recommended by the FDA in the latest guidance. Thesesamples were then aliquoted into 6 Nanotainer™ Tubes. Of these, threeNanotainer™ Tubes were loaded on ZEBOV GP antigen, field locationscartridges and processed directly on a SPU. The remaining 3 were spikedwith viral isolate to nominally have virus concentration at the LoD. Allsamples in Nanotainer™ Tubes were processed directly on a SPU. Theresults are summarized in Table N-6 (which presents a summary ofresponse of the ZEBOV GP antigen assay to known negative samples spikedwith interfering substances) and Table N-7 (which presents a Summary ofresponse of ZEBOV GP antigen assay to known positive samples (at 1×LoD)spiked with interfering substances).

TABLE N-6 Potential Interfering Substance Concentration Results(Detected X/3) Hemoglobin 20 g/dL 0/3 Bilirubin 25 mg/dL 0/3 Intralipids1500 mg/dL 0/3 Serum Protein 5 g/dL 0/3 HAMA (human anti-mouse 800 ng/ml0/3 antibody) Rheumatoid Factor 2000 IU/ml 2/3 Rheumatoid Factor 1000IU/ml 3/3 Rheumatoid Factor 500 IU/ml 3/3 Rheumatoid Factor 250 IU/ml0/3 Rheumatoid Factor 125 IU/ml 0/3

Interference from Rheumatoid factor was observed at high levels (>500IU/ml) which led to increase in observed signal and misclassification ofthe sample as positive. Therefore, false positive results may beobserved for subjects with very high levels of rheumatoid factor.According to a large study performed in Denmark (BMJ 2012; 345:e5244),250 IU/ml is in the 99.4^(th) percentile of the general population, and>95^(th) percentile for the 0.5-2% of the general population sufferingfrom Rheumatoid Arthritis, Sjogren's Syndrome and Lupus. This risk offalse positives is thus present in than 0.025%-0.1% of the generalpopulation.

TABLE N-7 Potential Interfering Substance Concentration Results(Detected X/3) Hemoglobin 20 g/dL 3/3 Bilirubin 25 mg/dL 3/3 Intralipids1500 mg/dL 3/3 Serum Protein 5 g/dL 3/3 Rheumatoid Factor 2000 IU/ml 3/3HAMA (human anti-mouse 800 ng/ml 3/3 antibody)

High Dose Hook Effect:

The data from titration of viral isolate summarized in Table N-2 did notshow a high dose hook effect. Due to the fact that viral antigenconcentration in the viral isolate could not be increased beyond theneat value, Applicant also used recombinant antigen spikes to verify thenon-existence of high dose hook effect. It should be noted that theZEBOV GP antigen assay uses sequential incubation of the sample and theAP-conjugated detection antibody with a wash in-between. Therefore theexistence of a high dose hook effect is extremely unlikely. The data aresummarized in Table N-8 below, which presents a summary ofconcentrations and COI measurements for high dose samples analyzed onZEBOV GP antigen assay.

TABLE N-8 Concentration of recombinant GP, uM COI Result 10.24 215 POS25.6 460 POS 64 672 POS 160 901 POS 400 1579 POS 1000 2161 POS 2500 2270POS

Mock Clinical Study:

In order to establish the performance characteristics of the ZEBOV GPantigen assay, a mock clinical study was conducted.

Samples from 100 individual subjects were analyzed in this study. The100 samples were divided into two sets: 50 samples were analyzedunspiked by three operators. Twenty-five samples were spiked with theviral isolate at the LoD and then analyzed in triplicate. The remainingtwenty-five samples were spiked at (4/3)×, 2×, and 4× the LoD(approximately 8 each) and then analyzed in triplicate (see Table N-9).Since the concentration of the neat viral isolate is only 40×LoD, anyspiking concentration greater than 4×LoD will lead to excessive dilutionof the whole blood matrix. Three operators processed the samples forthis mock clinical study in a blinded fashion. The results of the studyare presented in terms of percent agreement with expected results. TableN-9 presents a summary of concentrations used for contriving spikedsamples for the mock clinical study.

TABLE N-9 Final Spiked Concentra- tion ×10⁶ TCID₅₀/ml x LoD Number ofsubjects 0 0 50 6.25 1 25 8.33 4/3 9 12.5 2 8 25 4 8 Total 100

Study Protocol:

a) Capillary samples are collected in 3 Nanotainer™ Tubes from eachin-house subject. Venous samples from febrile patients are aliquotedinto three Nanotainer™ Tubes. b) Separately, 4 spiking stocks areprepared such that spiking 7 ul of the stock into a Nanotainer™ Tubewill yield final concentrations of 0, 1×LoD, 4/3×LoD, 2×LoD and 4×LoD.Spiking stocks are numbered 1 through 4, respectively. c) Each operatorwas provided with one Nanotainer™ Tube per subject. Each operator wasprovided cartridges labeled with the number of spiking solution. d)Operators spiked the Nanotainer™ Tubes inside a Biosafety Cabinet,inserted the Nanotainer™ Tube in ZEBOV GP antigen, field locationscartridges and process the sample directly on a SPU. e) In a blindedfashion, the results were analyzed by other members of the analyticalstaff and then matched with the key.

Data Analysis:

A) For each measurement, the following rules are applied to arrive atthe final result:

i. If COI≦1.0, the result is NEGATIVE

ii. If COI>1.0 AND COI≦1.6, the result is EQUIVOCAL

iii. If COI>1.6, the result is POSITIVE

B) For each subject, three measurements are available. If even one ofthese replicates is discrepant, the subject as a whole is considereddiscrepant.

C) The results on a per subject basis are compared to expected valuesand negative and positive percent agreements are calculated, along withexact 95% confidence intervals.

Out of the 100 samples analyzed, 15 were from febrile patients. Ofthese, 5 were run as negatives and 10 as spiked positives. For onesubject spiked at the LoD, one of the replicates tested as EQUIVOCAL,while the other two replicates tested as POSITIVE. This subject has beenconsidered as discrepant. Other subjects were 100% concordant. Thepercent agreements are summarized in Table N-10 below, which presents asummary of performance of the ZEBOV GP antigen assay in mock clinicalstudy.

TABLE N-10 Expected value Positive Equivocal Negative Total ZEBOV GPPositive 49 0 0 49 assay Equivocal 1 0 0 1 Negative 0 0 50 50 Total 50 050 100 95% Agreement Numerator/ Percent Confidence classificationDenominator Agreement Interval Negative Percent 50/50 100 92.9-100 Agreement Positive Percent 49/50 98 89.4-99.9 Agreement

Additionally, concentrations, amounts, and other numerical data may bepresented herein in a range format. It is to be understood that suchrange format is used merely for convenience and brevity and should beinterpreted flexibly to include not only the numerical values explicitlyrecited as the limits of the range, but also to include all theindividual numerical values or sub-ranges encompassed within that rangeas if each numerical value and sub-range is explicitly recited. Forexample, a size range of about 1 nm to about 200 nm should beinterpreted to include not only the explicitly recited limits of about 1nm and about 200 nm, but also to include individual sizes such as 2 nm,3 nm, 4 nm, and sub-ranges such as 10 nm to 50 nm, 20 nm to 100 nm, andother sub-ranges.

The publications discussed or cited herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.All publications mentioned herein are incorporated herein by referenceto disclose and describe the structures and/or methods in connectionwith which the publications are cited. For example, U.S. ProvisionalApplication Ser. No. 62/061,671 filed Oct. 8, 2014 is fully incorporatedherein by reference for all purposes.

While preferred embodiments of the present invention have been shown anddescribed herein, it may be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. Any feature, whetherpreferred or not, may be combined with any other feature, whetherpreferred or not. The appended claims are not to be interpreted asincluding means-plus-function limitations, unless such a limitation isexplicitly recited in a given claim using the phrase “means for.” Itshould be understood that as used in the description herein andthroughout the claims that follow, the meaning of “a,” “an,” and “the”includes plural reference unless the context clearly dictates otherwise.For example, a reference to “an assay” may refer to a single assay ormultiple assays. Also, as used in the description herein and throughoutthe claims that follow, the meaning of “in” includes “in” and “on”unless the context clearly dictates otherwise. Finally, as used in thedescription herein and throughout the claims that follow, the meaning of“or” includes both the conjunctive and disjunctive unless the contextexpressly dictates otherwise. Thus, the term “or” includes “and/or”unless the context expressly dictates otherwise.

This document contains material subject to copyright protection. Thecopyright owner (Applicant herein) has no objection to facsimilereproduction of the patent documents and disclosures, as they appear inthe US Patent and Trademark Office patent file or records, but otherwisereserves all copyright rights whatsoever. The following notice shallapply: Copyright 2014-2015 Thermos, Inc.

<160> NUMBER OF SEQ ID NOS: 6 <210> SEQ ID NO 1 <211> LENGTH: 18959<212> TYPE: DNA <213> ORGANISM: Zaire ebolavirus <400> SEQUENCE: 1cggacacaca aaaagaaaga agaattttta ggatcttttg tgtgcgaata actatgagga     60agattaataa ttttcctctc attgaaattt atatcggaat ttaaattgaa attgttactg    120taatcacacc tggtttgttt cagagccaca tcacaaagat agagaacaac ctaggtctcc    180gaagggagca agggcatcag tgtgctcagt tgaaaatccc ttgtcaacac ctaggtctta    240tcacatcaca agttccacct cagactctgc agggtgatcc aacaacctta atagaaacat    300tattgttaaa ggacagcatt agttcacagt caaacaagca agattgagaa ttaaccttgg    360ttttgaactt gaacacttag gggattgaag attcaacaac cctaaagctt ggggtaaaac    420attggaaata gttaaaagac aaattgctcg gaatcacaaa attccgagta tggattctcg    480tcctcagaaa atctggatgg cgccgagtct cactgaatct gacatggatt accacaagat    540cttgacagca ggtctgtccg ttcaacaggg gattgttcgg caaagagtca tcccagtgta    600tcaagtaaac aatcttgaag aaatttgcca acttatcata caggcctttg aagcaggtgt    660tgattttcaa gagagtgcgg acagtttcct tctcatgctt tgtcttcatc atgcgtacca    720gggagattac aaacttttct tggaaagtgg cgcagtcaag tatttggaag ggcacgggtt    780ccgttttgaa gtcaagaagc gtgatggagt gaagcgcctt gaggaattgc tgccagcagt    840atctagtgga aaaaacatta agagaacact tgctgccatg ccggaagagg agacaactga    900agctaatgcc ggtcagtttc tctcctttgc aagtctattc cttccgaaat tggtagtagg    960agaaaaggct tgccttgaga aggttcaaag gcaaattcaa gtacatgcag agcaaggact   1020gatacaatat ccaacagctt ggcaatcagt aggacacatg atggtgattt tccgtttgat   1080gcgaacaaat tttctgatca aatttctcct aatacaccaa gggatgcaca tggttgccgg   1140gcatgatgcc aacgatgctg tgatttcaaa ttcagtggct caagctcgtt tttcaggctt   1200attgattgtc aaaacagtac ttgatcatat cctacaaaag acagaacgag gagttcgtct   1260ccatcctctt gcaaggaccg ccaaggtaaa aaatgaggtg aactccttta aggctgcact   1320cagctccctg gccaagcatg gagagtatgc tcctttcgcc cgacttttga acctttctgg   1380agtaaataat cttgagcatg gtcttttccc tcaactatcg gcaattgcac tcggagtcgc   1440cacagcacac gggagtaccc tcgcaggagt aaatgttgga gaacagtatc aacaactcag   1500agaggctgcc actgaggctg agaagcaact ccaacaatat gcagagtctc gcgaacttga   1560ccatcttgga cttgatgatc aggaaaagaa aattcttatg aacttccatc agaaaaagaa   1620cgaaatcagc ttccagcaaa caaacgctat ggtaactcta agaaaagagc gcctggccaa   1680gctgacagaa gctatcactg ctgcgtcact gcccaaaaca agtggacatt acgatgatga   1740tgacgacatt ccctttccag gacccatcaa tgatgacgac aatcctggcc atcaagatga   1800tgatccgact gactcacagg atacgaccat tcccgatgtg gtggttgatc ctgatgatgg   1860aagctacggc gaataccaga gttactcgga aaacggcatg aatgcaccag atgacttggt   1920cctattcgat ctagacgagg acgacgagga cactaagcca gtgcctaata gatcgaccaa   1980gggtggacaa cagaagaaca gtcaaaaggg ccagcatata gagggcagac agacacaatt   2040caggccaatt caaaatgtcc caggccctca cagaacaatc caccacgcca gtgcgccact   2100cacggacaat gacagaagaa atgaaccctc cggctcaacc agccctcgca tgctgacacc   2160aattaacgaa gaggcagacc cactggacga tgccgacgac gagacgtcta gccttccgcc   2220cttggagtca gatgatgaag agcaggacag ggacggaact tccaaccgca cacccactgt   2280cgccccaccg gctcccgtat acagagatca ctctgaaaag aaagaactcc cgcaagacga   2340gcaacaagat caggaccaca ctcaagaggc caggaaccag gacagtgaca acacccagtc   2400agaacactcc cttgaggaga tgtatcgcca cattctaaga tcacaggggc catttgatgc   2460tgttttgtat tatcatatga tgaaggatga gcctgtagtt ttcagtacca gtgatggcaa   2520agagtacacg tatccagact cccttgaaga ggaatatcca ccatggctca ctgaaaaaga   2580ggctatgaat gaagagaata gatttgttac attggatggt caacaatttt attggccggt   2640gatgaatcac aagaataaat tcatggcaat cctgcaacat catcagtgaa tgagcatgga   2700acaatgggat gattcaaccg acaaatagct aacattaagt agtcaaggaa cgaaaacagg   2760aagaattttt gatgtctaag gtgtgaatta ttatcacaat aaaagtgatt cttatttttg   2820aatttaaagc tagcttatta ttactagccg tttttcaaag ttcaatttga gtcttaatgc   2880aaataggcgt taagccacag ttatagccat aattgtaact caatattcta actagcgatt   2940tatctaaatt aaattacatt atgcttttat aacttaccta ctagcctgcc caacatttac   3000acgatcgttt tataattaag aaaaaactaa tgatgaagat taaaaccttc atcatcctta   3060cgtcaattga attctctagc actcgaagct tattgtcttc aatgtaaaag aaaagctggt   3120ctaacaagat gacaactaga acaaagggca ggggccatac tgcggccacg actcaaaacg   3180acagaatgcc aggccctgag ctttcgggct ggatctctga gcagctaatg accggaagaa   3240ttcctgtaag cgacatcttc tgtgatattg agaacaatcc aggattatgc tacgcatccc   3300aaatgcaaca aacgaagcca aacccgaaga cgcgcaacag tcaaacccaa acggacccaa   3360tttgcaatca tagttttgag gaggtagtac aaacattggc ttcattggct actgttgtgc   3420aacaacaaac catcgcatca gaatcattag aacaacgcat tacgagtctt gagaatggtc   3480taaagccagt ttatgatatg gcaaaaacaa tctcctcatt gaacagggtt tgtgctgaga   3540tggttgcaaa atatgatctt ctggtgatga caaccggtcg ggcaacagca accgctgcgg   3600caactgaggc ttattgggcc gaacatggtc aaccaccacc tggaccatca ctttatgaag   3660aaagtgcgat tcggggtaag attgaatcta gagatgagac cgtccctcaa agtgttaggg   3720aggcattcaa caatctaaac agtaccactt cactaactga ggaaaatttt gggaaacctg   3780acatttcggc aaaggatttg agaaacatta tgtatgatca cttgcctggt tttggaactg   3840ctttccacca attagtacaa gtgatttgta aattgggaaa agatagcaac tcattggaca   3900tcattcatgc tgagttccag gccagcctgg ctgaaggaga ctctcctcaa tgtgccctaa   3960ttcaaattac aaaaagagtt ccaatcttcc aagatgctgc tccacctgtc atccacatcc   4020gctctcgagg tgacattccc cgagcttgcc agaaaagctt gcgtccagtc ccaccatcgc   4080ccaagattga tcgaggttgg gtatgtgttt ttcagcttca agatggtaaa acacttggac   4140tcaaaatttg agccaatctc ccttccctcc gaaagaggcg aataatagca gaggcttcaa   4200ctgctgaact atagggtacg ttacattaat gatacacttg tgagtatcag ccctggataa   4260tataagtcaa ttaaacgacc aagataaaat tgttcatatc tcgctagcag cttaaaatat   4320aaatgtaata ggagctatat ctctgacagt attataatca attgttatta agtaacccaa   4380accaaaagtg atgaagatta agaaaaacct acctcggctg agagagtgtt ttttcattaa   4440ccttcatctt gtaaacgttg agcaaaattg ttaaaaatat gaggcgggtt atattgccta   4500ctgctcctcc tgaatatatg gaggccatat accctgtcag gtcaaattca acaattgcta   4560gaggtggcaa cagcaataca ggcttcctga caccggagtc agtcaatggg gacactccat   4620cgaatccact caggccaatt gccgatgaca ccatcgacca tgccagccac acaccaggca   4680gtgtgtcatc agcattcatc cttgaagcta tggtgaatgt catatcgggc cccaaagtgc   4740taatgaagca aattccaatt tggcttcctc taggtgtcgc tgatcaaaag acctacagct   4800ttgactcaac tacggccgcc atcatgcttg cttcatacac tatcacccat ttcggcaagg   4860caaccaatcc acttgtcaga gtcaatcggc tgggtcctgg aatcccggat catcccctca   4920ggctcctgcg aattggaaac caggctttcc tccaggagtt cgttcttccg ccagtccaac   4980taccccagta tttcaccttt gatttgacag cactcaaact gatcacccaa ccactgcctg   5040ctgcaacatg gaccgatgac actccaacag gatcaaatgg agcgttgcgt ccaggaattt   5100catttcatcc aaaacttcgc cccattcttt tacccaacaa aagtgggaag aaggggaaca   5160gtgccgatct aacatctccg gagaaaatcc aagcaataat gacttcactc caggacttta   5220agatcgttcc aattgatcca accaaaaata tcatgggaat cgaagtgcca gaaactctgg   5280tccacaagct gaccggtaag aaggtgactt ctaaaaatgg acaaccaatc atccctgttc   5340ttttgccaaa gtacattggg ttggacccgg tggctccagg agacctcacc atggtaatca   5400cacaggattg tgacacgtgt cattctcctg caagtcttcc agctgtgatt gagaagtaat   5460tgcaataatt gactcagatc cagttttata gaatcttctc agggatagtg ataacatcta   5520tttagtaatc cgtccattag aggagacact tttaattgat caatatacta aaggtgcttt   5580acaccattgt cttttttctc tcctaaatgt agaacttaac aaaagactca taatatactt   5640gtttttaaag gattgattga tgaaagatca taactaataa cattacaaat aatcctacta   5700taatcaatac ggtgattcaa atgttaatct ttctcattgc acatactttt tgcccttatc   5760ctcaaattgc ctgcatgctt acatctgagg atagccagtg tgacttggat tggaaatgtg   5820gagaaaaaat cgggacccat ttctaggttg ttcacaatcc aagtacagac attgcccttc   5880taattaagaa aaaatcggcg atgaagatta agccgacagt gagcgtaatc ttcatctctc   5940ttagattatt tgttttccag agtaggggtc gtcaggtcct tttcaatcgt gtaaccaaaa   6000taaactccac tagaaggata ttgtggggca acaacacaat gggcgttaca ggaatattgc   6060agttacctcg tgatcgattc aagaggacat cattctttct ttgggtaatt atccttttcc   6120aaagaacatt ttccatccca cttggagtca tccacaatag cacattacag gttagtgatg   6180tcgacaaact agtttgtcgt gacaaactgt catccacaaa tcaattgaga tcagttggac   6240tgaatctcga agggaatgga gtggcaactg acgtgccatc tgcaactaaa agatggggct   6300tcaggtccgg tgtcccacca aaggtggtca attatgaagc tggtgaatgg gctgaaaact   6360gctacaatct tgaaatcaaa aaacctgacg ggagtgagtg tctaccagca gcgccagacg   6420ggattcgggg cttcccccgg tgccggtatg tgcacaaagt atcaggaacg ggaccgtgtg   6480ccggagactt tgccttccat aaagagggtg ctttcttcct gtatgatcga cttgcttcca   6540cagttatcta ccgaggaacg actttcgctg aaggtgtcgt tgcatttctg atactgcccc   6600aagctaagaa ggacttcttc agctcacacc ccttgagaga gccggtcaat gcaacggagg   6660acccgtctag tggctactat tctaccacaa ttagatatca ggctaccggt tttggaacca   6720atgagacaga gtacttgttc gaggttgaca atttgaccta cgtccaactt gaatcaagat   6780tcacaccaca gtttctgctc cagctgaatg agacaatata tacaagtggg aaaaggagca   6840ataccacggg aaaactaatt tggaaggtca accccgaaat tgatacaaca atcggggagt   6900gggccttctg ggaaactaaa aaaacctcac tagaaaaatt cgcagtgaag agttgtcttt   6960cacagttgta tcaaacggag ccaaaaacat cagtggtcag agtccggcgc gaacttcttc   7020cgacccaggg accaacacaa caactgaaga ccacaaaatc atggcttcag aaaattcctc   7080tgcaatggtt caagtgcaca gtcaaggaag ggaagctgca gtgtcgcatc taacaaccct   7140tgccacaatc tccacgagtc cccaatccct cacaaccaaa ccaggtccgg acaacagcac   7200ccataataca cccgtgtata aacttgacat ctctgaggca actcaagttg aacaacatca   7260ccgcagaaca gacaacgaca gcacagcctc cgacactccc tctgccacga ccgcagccgg   7320acccccaaaa gcagagaaca ccaacacgag caagagcact gacttcctgg accccgccac   7380cacaacaagt ccccaaaacc acagcgagac cgctggcaac aacaacactc atcaccaaga   7440taccggagaa gagagtgcca gcagcgggaa gctaggctta attaccaata ctattgctgg   7500agtcgcagga ctgatcacag gcgggagaag aactcgaaga gaagcaattg tcaatgctca   7560acccaaatgc aaccctaatt tacattactg gactactcag gatgaaggtg ctgcaatcgg   7620actggcctgg ataccatatt tcgggccagc agccgaggga atttacatag aggggctaat   7680gcacaatcaa gatggtttaa tctgtgggtt gagacagctg gccaacgaga cgactcaagc   7740tcttcaactg ttcctgagag ccacaactga gctacgcacc ttttcaatcc tcaaccgtaa   7800ggcaattgat ttcttgctgc agcgatgggg cggcacatgc cacattctgg gaccggactg   7860ctgtatcgaa ccacatgatt ggaccaagaa cataacagac aaaattgatc agattattca   7920tgattttgtt gataaaaccc ttccggacca gggggacaat gacaattggt ggacaggatg   7980gagacaatgg ataccggcag gtattggagt tacaggcgtt ataattgcag ttatcgcttt   8040attctgtata tgcaaatttg tcttttagtt tttcttcaga ttgcttcatg gaaaagctca   8100gcctcaaatc aatgaaacca ggatttaatt atatggatta cttgaatcta agattacttg   8160acaaatgata atataataca ctggagcttt aaacatagcc aatgtgattc taactccttt   8220aaactcacag ttaatcataa acaaggtttg acatcaatct agttatctct ttgagaatga   8280taaacttgat gaagattaag aaaaaggtaa tctttcgatt atctttaatc ttcatccttg   8340attctacaat catgacagtt gtctttagtg acaagggaaa gaagcctttt tattaagttg   8400taataatcag atctgcgaac cggtagagtt tagttgcaac ctaacacaca taaagcattg   8460gtcaaaaagt caatagaaat ttaaacagtg agtggagaca acttttaaat ggaagcttca   8520tatgagagag gacgcccacg agctgccaga cagcattcaa gggatggaca cgaccaccat   8580gttcgagcac gatcatcatc cagagagaat tatcgaggtg agtaccgtca atcaaggagc   8640gcctcacaag tgcgcgttcc tactgtattt cataagaaga gagttgaacc attaacagtt   8700cctccagcac ctaaagacat atgtccgacc ttgaaaaaag gatttttgtg tgacagtagt   8760ttttgcaaaa aagatcacca gttggagagt ttaactgata gggaattact cctactaatc   8820gcccgtaaga cttgtggatc agtagaacaa caattaaata taactgcacc caaggactcg   8880cgcttagcaa atccaacggc tgatgatttc cagcaagagg aaggtccaaa aattaccttg   8940ttgacactga tcaagacggc agaacactgg gcgagacaag acatcagaac catagaggat   9000tcaaaattaa gagcattgtt gactctatgt gctgtgatga cgaggaaatt ctcaaaatcc   9060cagctgagtc ttttatgtga gacacaccta aggcgcgagg ggcttgggca agatcaggca   9120gaacccgttc tcgaagtata tcaacgatta cacagtgata aaggaggcag ttttgaagct   9180gcactatggc aacaatggga ccgacaatcc ctaattatgt ttatcactgc attcttgaat   9240attgctctcc agttaccgtg tgaaagttct gctgtcgttg tttcagggtt aagaacattg   9300gttcctcaat cagataatga ggaagcttca accaacccgg ggacatgctc atggtctgat   9360gagggtaccc cttaataagg ctgactaaaa cactatataa ccttctactt gatcacaata   9420ctccgtatac ctatcatcat atatttaatc aagacgatat cctttaaaac ttattcagta   9480ctataatcac tctcgtttca aattaataag atgtgcatga ttgccctaat atatgaagag   9540gtatgataca accctaacag tgatcaaaga aaatcataat ctcgtatcgc tcgaaatata   9600acctgccaag catacctctt gcacaaagtg attcttgtac acaaataatg ttttactcta   9660caggaggtag caacgatcca tcccatcaaa aaataagtat ttcatgactt actaatgatc   9720tcttaaaata ttaagaaaaa ctgacggaac ataaattctt tatgcttcaa gctgtggagg   9780aggtgtttgg tattggctat tgttatatta caatcaataa caagcttgta aaaatattgt   9840tcttgtttca agaggtagat tgtgaccgga aatgctaaac taatgatgaa gattaatgcg   9900gaggtctgat aagaataaac cttattattc agattaggcc ccaagaggca ttcttcatct   9960ccttttagca aagtactatt tcagggtagt ccaattagtg gcacgtcttt tagctgtata  10020tcagtcgccc ctgagatacg ccacaaaagt gtctctaagc taaattggtc tgtacacatc  10080ccatacattg tattaggggc aataatatct aattgaactt agccgtttaa aatttagtgc  10140ataaatctgg gctaacacca ccaggtcaac tccattggct gaaaagaagc ttacctacaa  10200cgaacatcac tttgagcgcc ctcacaatta aaaaatagga acgtcgttcc aacaatcgag  10260cgcaaggttt caaggttgaa ctgagagtgt ctagacaaca aaatattgat actccagaca  10320ccaagcaaga cctgagaaaa aaccatggct aaagctacgg gacgatacaa tctaatatcg  10380cccaaaaagg acctggagaa aggggttgtc ttaagcgacc tctgtaactt cttagttagc  10440caaactattc aggggtggaa ggtttattgg gctggtattg agtttgatgt gactcacaaa  10500ggaatggccc tattgcatag actgaaaact aatgactttg cccctgcatg gtcaataaca  10560aggaatctct ttcctcattt atttcaaaat ccgaattcca caattgaatc accgctgtgg  10620gcattgagag tcatccttgc agcagggata caggaccagc tgattgacca gtctttgatt  10680gaacccttag caggagccct tggtctgatc tctgattggc tgctaacaac caacactaac  10740catttcaaca tgcgaacaca acgtgtcaag gaacaattga gcccaaaaat gctgtcgttg  10800attcgatcca atattctcaa gtttattaac aaattggatg ctctacatgt cgtgaactac  10860aacggattgt tgagcagtat tgaaattgga actcaaaatc atataatcat cataactcga  10920actaacatgg gttttctggt ggagctccaa gaacccgaca aatcggcaat gaaccgcatg  10980aagcctgggc cggcgaaatt ttccctcctt catgagtcca cactgaaagc atttacacaa  11040ggatcctcga cacgaatgca aagtttgatt cttgaattta atagctctct tgctatctaa  11100ctaaggtaga atacttcata ttgagctaac tcatatatgc tgactcaata gttatcttga  11160catctctgct ttcataatca gatatataag cataataaat aaatactcat atttcttgat  11220aatttgttta accacagata aatcctcact gtaagccagc ttccaagttg acacccttac  11280aaaaaccagg actcagaatc cctcaaacaa gagattccaa gacaacatca tagaattgct  11340ttattatatg aataagcatt ttatcaccag aaatcctata tactaaatgg ttaattgtaa  11400ctgaacccgc aggtcacatg tgttaggttt cacagattct atatattact aactctatac  11460tcgtaattaa cattagataa gtagattaag aaaaaagcct gaggaagatt aagaaaaact  11520gcttattggg tctttccgtg ttttagatga agcagttgaa attcttcctc ttgatattaa  11580atggctacac aacataccca atacccagac gctaggttat catcaccaat tgtattggac  11640caatgtgacc tagtcactag agcttgcggg ttatattcat catactccct taatccgcaa  11700ctacgcaact gtaaactccc gaaacatatc taccgtttga aatacgatgt aactgttacc  11760aagttcttga gtgatgtacc agtggcgaca ttgcccatag atttcatagt cccagttctt  11820ctcaaggcac tgtcaggcaa tggattctgt cctgttgagc cgcggtgcca acagttctta  11880gatgaaatca ttaagtacac aatgcaagat gctctcttct tgaaatatta tctcaaaaat  11940gtgggtgctc aagaagactg tgttgatgaa cactttcaag agaaaatctt atcttcaatt  12000cagggcaatg aatttttaca tcaaatgttt ttctggtatg atctggctat tttaactcga  12060aggggtagat taaatcgagg aaactctaga tcaacatggt ttgttcatga tgatttaata  12120gacatcttag gctatgggga ctatgttttt tggaagatcc caatttcaat gttaccactg  12180aacacacaag gaatccccca tgctgctatg gactggtatc aggcatcagt attcaaagaa  12240gcggttcaag ggcatacaca cattgtttct gtttctactg ccgacgtctt gataatgtgc  12300aaagatttaa ttacatgtcg attcaacaca actctaatct caaaaatagc agagattgag  12360gatccagttt gttctgatta tcccaatttt aagattgtgt ctatgcttta ccagagcgga  12420gattacttac tctccatatt agggtctgat gggtataaaa ttattaagtt cctcgaacca  12480ttgtgcttgg ccaaaattca attatgctca aagtacactg agaggaaggg ccgattctta  12540acacaaatgc atttagctgt aaatcacacc ctagaagaaa ttacagaaat gcgtgcacta  12600aagccttcac aggctcaaaa gatccgtgaa ttccatagaa cattgataag gctggagatg  12660acgccacaac aactttgtga gctattttcc attcaaaaac actgggggca tcctgtgcta  12720catagtgaaa cagcaatcca aaaagttaaa aaacatgcta cggtgctaaa agcattacgc  12780cctatagtga ttttcgagac atactgtgtt tttaaatata gtattgccaa acattatttt  12840gatagtcaag gatcttggta cagtgttact tcagatagga atctaacacc gggtcttaat  12900tcttatatca aaagaaatca attccctccg ttgccaatga ttaaagaact actatgggaa  12960ttttaccacc ttgaccaccc tccacttttc tcaaccaaaa ttattagtga cttaagtatt  13020tttataaaag acagagctac cgcagtagaa aggacatgct gggatgcagt attcgagcct  13080aatgttctag gatataatcc acctcacaaa tttagtacta aacgtgtacc ggaacaattt  13140ttagagcaag aaaacttttc tattgagaat gttctttcct acgcacaaaa actcgagtat  13200ctactaccac aatatcggaa cttttctttc tcattgaaag agaaagagtt gaatgtaggt  13260agaaccttcg gaaaattgcc ttatccgact cgcaatgttc aaacactttg tgaagctctg  13320ttagctgatg gtcttgctaa agcatttcct agcaatatga tggtagttac ggaacgtgag  13380caaaaagaaa gcttattgca tcaagcatca tggcaccaca caagtgatga ttttggtgaa  13440catgccacag ttagagggag tagctttgta actgatttag agaaatacaa tcttgcattt  13500agatatgagt ttacagcacc ttttatagaa tattgcaacc gttgctatgg tgttaagaat  13560gtttttaatt ggatgcatta tacaatccca cagtgttata tgcatgtcag tgattattat  13620aatccaccac ataacctcac actggagaat cgagacaacc cccccgaagg gcctagttca  13680tacaggggtc atatgggagg gattgaagga ctgcaacaaa aactctggac aagtatttca  13740tgtgctcaaa tttctttagt tgaaattaag actggtttta agttacgctc agctgtgatg  13800ggtgacaatc agtgcattac tgttttatca gtcttcccct tagagactga cgcagacgag  13860caggaacaga gcgccgaaga caatgcagcg agggtggccg ccagcctagc aaaagttaca  13920agtgcctgtg gaatcttttt aaaacctgat gaaacatttg tacattcagg ttttatctat  13980tttggaaaaa aacaatattt gaatggggtc caattgcctc agtcccttaa aacggctgca  14040agaatggcac cattgtctga tgcaattttt gatgatcttc aagggaccct ggctagtata  14100ggcactgctt ttgagcgatc catctctgag acacgacata tctttccttg caggataacc  14160gcagctttcc atacgttttt ttcggtgaga atcttgcaat atcatcatct cgggttcaat  14220aaaggttttg accttggaca gttaacactc ggcaaacctc tggatttcgg aacaatatca  14280ttggcactag cggtaccgca ggtgcttgga gggttatcct tcttgaatcc tgagaaatgt  14340ttctaccgga atctaggaga tccagttacc tcaggcttat tccagttaaa aacttatctc  14400cgaatgattg agatggatga tttattctta cctttaattg cgaagaaccc tgggaactgc  14460actgccattg actttgtgct aaatcctagc ggattaaatg tccctgggtc gcaagactta  14520acttcatttc tgcgccagat tgtacgcagg accatcaccc taagtgcgaa aaacaaactt  14580attaatacct tatttcatgc gtcagctgac ttcgaagacg aaatggtttg taaatggcta  14640ttatcatcaa ctcctgttat gagtcgtttt gcggccgata tcttttcacg cacgccgagc  14700gggaagcgat tgcaaattct aggatacctg gaaggaacac gcacattatt agcctctaag  14760atcatcaaca ataatacaga gacaccggtt ttggacagac tgaggaaaat aacattgcaa  14820aggtggagcc tatggtttag ttatcttgat cattgtgata atatcctggc ggaggcttta  14880acccaaataa cttgcacagt tgatttagca cagattctga gggaatattc atgggctcat  14940attttagagg gaagacctct tattggagcc acactcccat gtatgattga gcaattcaaa  15000gtgttttggc tgaaacccta cgaacaatgt ccgcagtgtt caaatgcaaa gcaaccaggt  15060gggaaaccat tcgtgtcagt ggcagtcaag aaacatattg ttagtgcatg gccgaacgca  15120tcccgaataa gctggactat cggggatgga atcccataca ttggatcaag gacagaagat  15180aagataggac aacctgctat taaaccaaaa tgtccttccg cagccttaag agaggccatt  15240gaattggcgt cccgtttaac atgggtaact caaggcagtt cgaacagtga cttgctaata  15300aaaccatttt tggaagcacg agtaaattta agtgttcaag aaatacttca aatgacccct  15360tcacattact caggaaatat tgttcacagg tacaacgatc aatacagtcc tcattctttc  15420atggccaatc gtatgagtaa ttcagcaacg cgattgattg tttctacaaa cactttaggt  15480gagttttcag gaggtggcca gtctgcacgc gacagcaata ttattttcca gaatgttata  15540aattatgcag ttgcactgtt cgatattaaa tttagaaaca ctgaggctac agatatccaa  15600tataatcgtg ctcaccttca tctaactaag tgttgcaccc gggaagtacc agctcagtat  15660ttaacataca catctacatt ggatttagat ttaacaagat accgagaaaa cgaattgatt  15720tatgacagta atcctctaaa aggaggactc aattgcaata tctcattcga taatccattt  15780ttccaaggta aacggctgaa cattatagaa gatgatctta ttcgactgcc tcacttatct  15840ggatgggagc tagccaagac catcatgcaa tcaattattt cagatagcaa caattcatct  15900acagacccaa ttagcagtgg agaaacaaga tcattcacta cccatttctt aacttatccc  15960aagataggac ttctgtacag ttttggggcc tttgtaagtt attatcttgg caatacaatt  16020cttcggacta agaaattaac acttgacaat tttttatatt acttaactac tcaaattcat  16080aatctaccac atcgctcatt gcgaatactt aagccaacat tcaaacatgc aagcgttatg  16140tcacggttaa tgagtattga tcctcatttt tctatttaca taggcggtgc tgcaggtgac  16200agaggactct cagatgcggc caggttattt ttgagaacgt ccatttcatc ttttcttaca  16260tttgtaaaag aatggataat taatcgcgga acaattgtcc ctttatggat agtatatccg  16320ctagagggtc aaaacccaac acctgtgaat aattttctct atcagatcgt agaactgctg  16380gtgcatgatt catcaagaca acaggctttt aaaactacca taagtgatca tgtacatcct  16440cacgacaatc ttgtttacac atgtaagagt acagccagca atttcttcca tgcatcattg  16500gcgtactgga ggagcagaca cagaaacagc aaccgaaaat acttggcaag agactcttca  16560actggatcaa gcacaaacaa cagtgatggt catattgaga gaagtcaaga acaaaccacc  16620agagatccac atgatggcac tgaacggaat ctagtcctac aaatgagcca tgaaataaaa  16680agaacgacaa ttccacaaga aaacacgcac cagggtccgt cgttccagtc ctttctaagt  16740gactctgctt gtggtacagc aaatccaaaa ctaaatttcg atcgatcgag acacaatgtg  16800aaatttcagg atcataactc ggcatccaag agggaaggtc atcaaataat ctcacaccgt  16860ctagtcctac ctttctttac attatctcaa gggacacgcc aattaacgtc atccaatgag  16920tcacaaaccc aagacgagat atcaaagtac ttacggcaat tgagatccgt cattgatacc  16980acagtttatt gtagatttac cggtatagtc tcgtccatgc attacaaact tgatgaggtc  17040ctttgggaaa tagagagttt caagtcggct gtgacgctag cagagggaga aggtgctggt  17100gccttactat tgattcagaa ataccaagtt aagaccttat ttttcaacac gctagctact  17160gagtccagta tagagtcaga aatagtatca ggaatgacta ctcctaggat gcttctacct  17220gttatgtcaa aattccataa tgaccaaatt gagattattc ttaacaactc agcaagccaa  17280ataacagaca taacaaatcc tacttggttt aaagaccaaa gagcaaggct acctaagcaa  17340gtcgaggtta taaccatgga tgcagagaca acagagaata taaacagatc gaaattgtac  17400gaagctgtat ataaattgat cttacaccat attgatccta gcgtattgaa agcagtggtc  17460cttaaagtct ttctaagtga tactgagggt atgttatggc taaatgataa tttagccccg  17520ttttttgcca ctggttattt aattaagcca ataacgtcaa gtgctagatc tagtgagtgg  17580tatctttgtc tgacgaactt cttatcaact acacgtaaga tgccacacca aaaccatctc  17640agttgtaaac aggtaatact tacggcattg caactgcaaa ttcaacgaag cccatactgg  17700ctaagtcatt taactcagta tgctgactgt gagttacatt taagttatat ccgccttggt  17760tttccatcat tagagaaagt actataccac aggtataacc tcgtcgattc aaaaagaggt  17820ccactagtct ctatcactca gcacttagca catcttagag cagagattcg agaattaact  17880aatgattata atcaacagcg acaaagtcgg actcaaacat atcactttat tcgtactgca  17940aaaggacgaa tcacaaaact agtcaatgat tatttaaaat tctttcttat tgtgcaagca  18000ttaaaacata atgggacatg gcaagctgag tttaagaaat taccagagtt gattagtgtg  18060tgcaataggt tctaccatat tagagattgc aattgtgaag aacgtttctt agttcaaacc  18120ttatatttac atagaatgca ggattctgaa gttaagctta tcgaaaggct gacagggctt  18180ctgagtttat ttccggatgg tctctacagg tttgattgaa ttaccgtgca tagtatcctg  18240atacttgcaa aggttggtta ttaacataca gattataaaa aactcataaa ttgctctcat  18300acatcatatt gatctaatct caataaacaa ctatttaaat aacgaaagga gtccctatat  18360tatatactat atttagcctc tctccctgcg tgataatcaa aaaattcaca atgcagcatg  18420tgtgacatat tactgccgca atgaatttaa cgcaacataa taaactctgc actctttata  18480attaagcttt aacgaaaggt ctgggctcat attgttattg atataataat gttgtatcaa  18540tatcctgtca gatggaatag tgttttggtt gataacacaa cttcttaaaa caaaattgat  18600ctttaagatt aagtttttta taattatcat tactttaatt tgtcgtttta aaaacggtga  18660tagccttaat ctttgtgtaa aataagagat taggtgtaat aaccttaaca tttttgtcta  18720gtaagctact atttcataca gaatgataaa attaaaagaa aaggcaggac tgtaaaatca  18780gaaatacctt ctttacaata tagcagacta gataataatc ttcgtgttaa tgataattaa  18840gacattgacc acgctcatca gaaggctcgc cagaataaac gttgcaaaaa ggattcctgg  18900aaaaatggtc gcacacaaaa atttaaaaat aaatctattt cttctttttt gtgtgtcca   18959<210> SEQ ID NO 2 <211> LENGTH: 3021 <212> TYPE: DNA<213> ORGANISM: Zaire ebolavirus <400> SEQUENCE: 2cggacacaca aaaagaaaga agaattttta ggatcttttg tgtgcgaata actatgagga     60agattaataa ttttcctctc attgaaattt atatcggaat ttaaattgaa attgttactg    120taatcacacc tggtttgttt cagagccaca tcacaaagat agagaacaac ctaggtctcc    180gaagggagca agggcatcag tgtgctcagt tgaaaatccc ttgtcaacac ctaggtctta    240tcacatcaca agttccacct cagactctgc agggtgatcc aacaacctta atagaaacat    300tattgttaaa ggacagcatt agttcacagt caaacaagca agattgagaa ttaaccttgg    360ttttgaactt gaacacttag gggattgaag attcaacaac cctaaagctt ggggtaaaac    420attggaaata gttaaaagac aaattgctcg gaatcacaaa attccgagta tggattctcg    480tcctcagaaa atctggatgg cgccgagtct cactgaatct gacatggatt accacaagat    540cttgacagca ggtctgtccg ttcaacaggg gattgttcgg caaagagtca tcccagtgta    600tcaagtaaac aatcttgaag aaatttgcca acttatcata caggcctttg aagcaggtgt    660tgattttcaa gagagtgcgg acagtttcct tctcatgctt tgtcttcatc atgcgtacca    720gggagattac aaacttttct tggaaagtgg cgcagtcaag tatttggaag ggcacgggtt    780ccgttttgaa gtcaagaagc gtgatggagt gaagcgcctt gaggaattgc tgccagcagt    840atctagtgga aaaaacatta agagaacact tgctgccatg ccggaagagg agacaactga    900agctaatgcc ggtcagtttc tctcctttgc aagtctattc cttccgaaat tggtagtagg    960agaaaaggct tgcttgagga aggttcaaag gcaaattcaa gtacatgcag agcaaggact   1020gatacaatat ccaacagctt ggcaatcagt aggacacatg atggtgattt tccgtttgat   1080gcgaacaaat tttctgatca aatttctcct aatacaccaa gggatgcaca tggttgccgg   1140gcatgatgcc aacgatgctg tgatttcaaa ttcagtggct caagctcgtt tttcaggctt   1200attgattgtc aaaacagtac ttgatcatat cctacaaaag acagaacgag gagttcgtct   1260ccatcctctt gcaaggaccg ccaaggtaaa aaatgaggtg aactccctta aggctgcact   1320cagctccctg gccaagcatg gagagtatgc tcctttcgcc cgacttttga acctttctgg   1380agtaaataat cttgagcatg gtcttttccc tcaactatcg gcaattgcac tcggagtcgc   1440cacagcacac gggagtaccc tcgcaggagt aaatgttgga gaacagtatc aacaactcag   1500agaggctgcc actgaggctg agaagcaact ccaacaatat gcagagtctc gcgaacttga   1560ccatcttgga cttgatgatc aggaaaagaa aattcttatg aacttccatc agaaaaagaa   1620cgaaatcagc ttccagcaaa caaacgctat ggtaactcta agaaaagagc gcctggccaa   1680gctgacagaa gctatcactg ctgcgtcact gcccaaaaca agtggacatt acgatgatga   1740tgacgacatt ccctttccag gacccatcaa tgatgacgac aatcctggcc atcaagatga   1800tgatccgact gactcacagg atacgaccat tcccgatgtg gtggttgatc ccgatgatgg   1860aagctacggc gaataccaga gttactcgga aaacggcatg aatgcaccag atgacttggt   1920cctattcgat ctagacgagg acgacgagga cactaagcca gtgcctaata gatcgaccaa   1980gggtggacaa cagaagaaca gtcaaaaggg ccagcatata gagggcagac agacacaatc   2040caggccaatt caaaatgtcc caggccctca cagaacaatc caccacgcca gtgcgccact   2100cacggacaat gacagaagaa atgaaccctc cggctcaacc agccctcgca tgctgacacc   2160aattaacgaa gaggcagacc cactggacga tgccgacgac gagacgtcta gccttccgcc   2220cttggagtca gatgatgaag agcaggacag ggacggaact tccaaccgca cacccactgt   2280cgccccaccg gctcccgtat acagagatca ctctgaaaag aaagaactcc cgcaagacga   2340gcaacaagat caggaccaca ctcaagaggc caggaaccag gacagtgaca acacccagtc   2400agaacactct tttgaggaga tgtatcgcca cattctaaga tcacaggggc catttgatgc   2460tgttttgtat tatcatatga tgaaggatga gcctgtagtt ttcagtacca gtgatggcaa   2520agagtacacg tatccagact cccttgaaga ggaatatcca ccatggctca ctgaaaaaga   2580ggctatgaat gaagagaata gatttgttac attggatggt caacaatttt attggccggt   2640gatgaatcac aagaataaat tcatggcaat cctgcaacat catcagtgaa tgagcatgga   2700acaatgggat gattcaaccg acaaatagct aacattaagt agtcaaggaa cgaaaacagg   2760aagaattttt gatgtctaag gtgtgaatta ttatcacaat aaaagtgatt cttatttttg   2820aatttaaagc tagccttatt attactagcc gtttttcaaa gttcaatttg agtcttaatg   2880caaataggcg ttaagccaca gttatagcca taattgtaac tcaatattct aactagcgat   2940ttatctaaat taaattacat tatgctttta taacttacct actagcctgc ccaacattta   3000cacgatcgtt ttataattaa g                                             3021<210> SEQ ID NO 3 <211> LENGTH: 2408 <212> TYPE: DNA<213> ORGANISM: Zaire ebolavirus <400> SEQUENCE: 3gcgatgaaga ttaagccgac agtgagcgta atcttcatct ctcttagatt atttgttttc     60cagagtaggg gtcgtcaggt ccttttcaat cgtgtaacca aaataaactc cactagaagg    120atattgtggg gcaacaacac aatgggcgtt acaggaatat tgcagttacc tcgtgatcga    180ttcaagagga catcattctt tctttgggta attatccttt tccaaagaac attttccatc    240ccacttggag tcatccacaa tagcacatta caggttagtg atgtcgacaa actagtttgt    300cgtgacaaac tgtcatccac aaatcaattg agatcagttg gactgaatct cgaagggaat    360ggagtggcaa ctgacgtgcc atctgcaact aaaagatggg gcttcaggtc cggtgtccca    420ccaaaggtgg tcaattatga agctggtgaa tgggctgaaa actgctacaa tcttgaaatc    480aaaaaacctg acgggagtga gtgtctacca gcagcgccag acgggattcg gggcttcccc    540cggtgccggt atgtgcacaa agtatcagga acgggaccgt gtgccggaga ctttgccttc    600cataaagagg gtgctttctt cctgtatgat cgacttgctt ccacagttat ctaccgagga    660acgactttcg ctgaaggtgt cgttgcattt ctgatactgc cccaagctaa gaaggacttc    720ttcagctcac accccttgag agagccggtc aatgcaacgg aggacccgtc tagtggctac    780tattctacca caattagata tcaggctacc ggttttggaa ccaatgagac agagtacttg    840ttcgaggttg acaatttgac ctacgtccaa cttgaatcaa gattcacacc acagtttctg    900ctccagctga atgagacaat atatacaagt gggaaaagga gcaataccac gggaaaacta    960atttggaagg tcaaccccga aattgataca acaatcgggg agtgggcctt ctgggaaact   1020aaaaaaacct cactagaaaa attcgcagtg aagagttgtc tttcacagtt gtatcaaacg   1080gagccaaaaa catcagtggt cagagtccgg cgcgaacttc ttccgaccca gggaccaaca   1140caacaactga agaccacaaa atcatggctt cagaaaattc ctctgcaatg gttcaagtgc   1200acagtcaagg aagggaagct gcagtgtcgc atctaacaac ccttgccaca atctccacga   1260gtccccaatc cctcacaacc aaaccaggtc cggacaacag cacccataat acacccgtgt   1320ataaacttga catctctgag gcaactcaag ttgaacaaca tcaccgcaga acagacaacg   1380acagcacagc ctccgacact ccctctgcca cgaccgcagc cggaccccca aaagcagaga   1440acaccaacac gagcaagagc actgacttcc tggaccccgc caccacaaca agtccccaaa   1500accacagcga gaccgctggc aacaacaaca ctcatcacca agataccgga gaagagagtg   1560ccagcagcgg gaagctaggc ttaattacca atactattgc tggagtcgca ggactgatca   1620caggcgggag aagaactcga agagaagcaa ttgtcaatgc tcaacccaaa tgcaacccta   1680atttacatta ctggactact caggatgaag gtgctgcaat cggactggcc tggataccat   1740atttcgggcc agcagccgag ggaatttaca tagaggggct aatgcacaat caagatggtt   1800taatctgtgg gttgagacag ctggccaacg agacgactca agctcttcaa ctgttcctga   1860gagccacaac tgagctacgc accttttcaa tcctcaaccg taaggcaatt gatttcttgc   1920tgcagcgatg gggcggcaca tgccacattc tgggaccgga ctgctgtatc gaaccacatg   1980attggaccaa gaacataaca gacaaaattg atcagattat tcatgatttt gttgataaaa   2040cccttccgga ccagggggac aatgacaatt ggtggacagg atggagacaa tggataccgg   2100caggtattgg agttacaggc gttataattg cagttatcgc tttattctgt atatgcaaat   2160ttgtctttta gtttttcttc agattgcttc atggaaaagc tcagcctcaa atcaatgaaa   2220ccaggattta attatatgga ttacttgaat ctaagattac ttgacaaatg ataatataat   2280acactggagc tttaaacata gccaatgtga ttctaactcc tttaaactca cagttaatca   2340taaacaaggt ttgacatcaa tctagttatc tctttgagaa tgataaactt gatgaagatt   2400aagaaaaa                                                            2408<210> SEQ ID NO 4 <211> LENGTH: 739 <212> TYPE: PRT<213> ORGANISM: Zaire ebolavirus <400> SEQUENCE: 4Met Asp Ser Arg Pro Gln Lys Ile Trp Met Ala Pro Ser Leu Thr Glu 1               5                   10                  15      Ser Asp Met Asp Tyr His Lys Ile Leu Thr Ala Gly Leu Ser Val Gln             20                  25                  30          Gln Gly Ile Val Arg Gln Arg Val Ile Pro Val Tyr Gln Val Asn Asn         35                  40                  45              Leu Glu Glu Ile Cys Gln Leu Ile Ile Gln Ala Phe Glu Ala Gly Val     50                  55                  60                  Asp Phe Gln Glu Ser Ala Asp Ser Phe Leu Leu Met Leu Cys Leu His 65                  70                  75                  80  His Ala Tyr Gln Gly Asp Tyr Lys Leu Phe Leu Glu Ser Gly Ala Val                 85                  90                  95      Lys Tyr Leu Glu Gly His Gly Phe Arg Phe Glu Val Lys Lys Arg Asp             100                 105                 110         Gly Val Lys Arg Leu Glu Glu Leu Leu Pro Ala Val Ser Ser Gly Lys         115                 120                 125             Asn Ile Lys Arg Thr Leu Ala Ala Met Pro Glu Glu Glu Thr Thr Glu     130                 135                 140                 Ala Asn Ala Gly Gln Phe Leu Ser Phe Ala Ser Leu Phe Leu Pro Lys 145                 150                 155                 160 Leu Val Val Gly Glu Lys Ala Cys Leu Arg Lys Val Gln Arg Gln Ile                 165                 170                 175     Gln Val His Ala Glu Gln Gly Leu Ile Gln Tyr Pro Thr Ala Trp Gln             180                 185                 190         Ser Val Gly His Met Met Val Ile Phe Arg Leu Met Arg Thr Asn Phe         195                 200                 205             Leu Ile Lys Phe Leu Leu Ile His Gln Gly Met His Met Val Ala Gly     210                 215                 220                 His Asp Ala Asn Asp Ala Val Ile Ser Asn Ser Val Ala Gln Ala Arg 225                 230                 235                 240 Phe Ser Gly Leu Leu Ile Val Lys Thr Val Leu Asp His Ile Leu Gln                 245                 250                 255     Lys Thr Glu Arg Gly Val Arg Leu His Pro Leu Ala Arg Thr Ala Lys             260                 265                 270         Val Lys Asn Glu Val Asn Ser Leu Lys Ala Ala Leu Ser Ser Leu Ala         275                 280                 285             Lys His Gly Glu Tyr Ala Pro Phe Ala Arg Leu Leu Asn Leu Ser Gly     290                 295                 300                 Val Asn Asn Leu Glu His Gly Leu Phe Pro Gln Leu Ser Ala Ile Ala 305                 310                 315                 320 Leu Gly Val Ala Thr Ala His Gly Ser Thr Leu Ala Gly Val Asn Val                 325                 330                 335     Gly Glu Gln Tyr Gln Gln Leu Arg Glu Ala Ala Thr Glu Ala Glu Lys             340                 345                 350         Gln Leu Gln Gln Tyr Ala Glu Ser Arg Glu Leu Asp His Leu Gly Leu         355                 360                 365             Asp Asp Gln Glu Lys Lys Ile Leu Met Asn Phe His Gln Lys Lys Asn     370                 375                 380                 Glu Ile Ser Phe Gln Gln Thr Asn Ala Met Val Thr Leu Arg Lys Glu 385                 390                 395                 400 Arg Leu Ala Lys Leu Thr Glu Ala Ile Thr Ala Ala Ser Leu Pro Lys                 405                 410                 415     Thr Ser Gly His Tyr Asp Asp Asp Asp Asp Ile Pro Phe Pro Gly Pro             420                 425                 430         Ile Asn Asp Asp Asp Asn Pro Gly His Gln Asp Asp Asp Pro Thr Asp         435                 440                 445             Ser Gln Asp Thr Thr Ile Pro Asp Val Val Val Asp Pro Asp Asp Gly     450                 455                 460                 Ser Tyr Gly Glu Tyr Gln Ser Tyr Ser Glu Asn Gly Met Asn Ala Pro 465                 470                 475                 480 Asp Asp Leu Val Leu Phe Asp Leu Asp Glu Asp Asp Glu Asp Thr Lys                 485                 490                 495     Pro Val Pro Asn Arg Ser Thr Lys Gly Gly Gln Gln Lys Asn Ser Gln             500                 505                 510         Lys Gly Gln His Ile Glu Gly Arg Gln Thr Gln Ser Arg Pro Ile Gln         515                 520                 525             Asn Val Pro Gly Pro His Arg Thr Ile His His Ala Ser Ala Pro Leu     530                 535                 540                 Thr Asp Asn Asp Arg Arg Asn Glu Pro Ser Gly Ser Thr Ser Pro Arg 545                 550                 555                 560 Met Leu Thr Pro Ile Asn Glu Glu Ala Asp Pro Leu Asp Asp Ala Asp                 565                 570                 575     Asp Glu Thr Ser Ser Leu Pro Pro Leu Glu Ser Asp Asp Glu Glu Gln             580                 585                 590         Asp Arg Asp Gly Thr Ser Asn Arg Thr Pro Thr Val Ala Pro Pro Ala         595                 600                 605             Pro Val Tyr Arg Asp His Ser Glu Lys Lys Glu Leu Pro Gln Asp Glu     610                 615                 620                 Gln Gln Asp Gln Asp His Thr Gln Glu Ala Arg Asn Gln Asp Ser Asp 625                 630                 635                 640 Asn Thr Gln Ser Glu His Ser Phe Glu Glu Met Tyr Arg His Ile Leu                 645                 650                 655     Arg Ser Gln Gly Pro Phe Asp Ala Val Leu Tyr Tyr His Met Met Lys             660                 665                 670         Asp Glu Pro Val Val Phe Ser Thr Ser Asp Gly Lys Glu Tyr Thr Tyr         675                 680                 685             Pro Asp Ser Leu Glu Glu Glu Tyr Pro Pro Trp Leu Thr Glu Lys Glu     690                 695                 700                 Ala Met Asn Glu Glu Asn Arg Phe Val Thr Leu Asp Gly Gln Gln Phe 705                 710                 715                 720 Tyr Trp Pro Val Met Asn His Lys Asn Lys Phe Met Ala Ile Leu Gln                 725                 730                 735     His His Gln  <210> SEQ ID NO 5 <211> LENGTH: 739 <212> TYPE: PRT<213> ORGANISM: Zaire ebolavirus <400> SEQUENCE: 5Met Asp Ser Arg Pro Gln Lys Ile Trp Met Ala Pro Ser Leu Thr Glu 1               5                   10                  15      Ser Asp Met Asp Tyr His Lys Ile Leu Thr Ala Gly Leu Ser Val Gln             20                  25                  30          Gln Gly Ile Val Arg Gln Arg Val Ile Pro Val Tyr Gln Val Asn Asn         35                  40                  45              Leu Glu Glu Ile Cys Gln Leu Ile Ile Gln Ala Phe Glu Ala Gly Val     50                  55                  60                  Asp Phe Gln Glu Ser Ala Asp Ser Phe Leu Leu Met Leu Cys Leu His 65                  70                  75                  80  His Ala Tyr Gln Gly Asp Tyr Lys Leu Phe Leu Glu Ser Gly Ala Val                 85                  90                  95      Lys Tyr Leu Glu Gly His Gly Phe Arg Phe Glu Val Lys Lys Arg Asp             100                 105                 110         Gly Val Lys Arg Leu Glu Glu Leu Leu Pro Ala Val Ser Ser Gly Lys         115                 120                 125             Asn Ile Lys Arg Thr Leu Ala Ala Met Pro Glu Glu Glu Thr Thr Glu     130                 135                 140                 Ala Asn Ala Gly Gln Phe Leu Ser Phe Ala Ser Leu Phe Leu Pro Lys 145                 150                 155                 160 Leu Val Val Gly Glu Lys Ala Cys Leu Glu Lys Val Gln Arg Gln Ile                 165                 170                 175     Gln Val His Ala Glu Gln Gly Leu Ile Gln Tyr Pro Thr Ala Trp Gln             180                 185                 190         Ser Val Gly His Met Met Val Ile Phe Arg Leu Met Arg Thr Asn Phe         195                 200                 205             Leu Ile Lys Phe Leu Leu Ile His Gln Gly Met His Met Val Ala Gly     210                 215                 220                 His Asp Ala Asn Asp Ala Val Ile Ser Asn Ser Val Ala Gln Ala Arg 225                 230                 235                 240 Phe Ser Gly Leu Leu Ile Val Lys Thr Val Leu Asp His Ile Leu Gln                 245                 250                 255     Lys Thr Glu Arg Gly Val Arg Leu His Pro Leu Ala Arg Thr Ala Lys             260                 265                 270         Val Lys Asn Glu Val Asn Ser Phe Lys Ala Ala Leu Ser Ser Leu Ala         275                 280                 285             Lys His Gly Glu Tyr Ala Pro Phe Ala Arg Leu Leu Asn Leu Ser Gly     290                 295                 300                 Val Asn Asn Leu Glu His Gly Leu Phe Pro Gln Leu Ser Ala Ile Ala 305                 310                 315                 320 Leu Gly Val Ala Thr Ala His Gly Ser Thr Leu Ala Gly Val Asn Val                 325                 330                 335     Gly Glu Gln Tyr Gln Gln Leu Arg Glu Ala Ala Thr Glu Ala Glu Lys             340                 345                 350         Gln Leu Gln Gln Tyr Ala Glu Ser Arg Glu Leu Asp His Leu Gly Leu         355                 360                 365             Asp Asp Gln Glu Lys Lys Ile Leu Met Asn Phe His Gln Lys Lys Asn     370                 375                 380                 Glu Ile Ser Phe Gln Gln Thr Asn Ala Met Val Thr Leu Arg Lys Glu 385                 390                 395                 400 Arg Leu Ala Lys Leu Thr Glu Ala Ile Thr Ala Ala Ser Leu Pro Lys                 405                 410                 415     Thr Ser Gly His Tyr Asp Asp Asp Asp Asp Ile Pro Phe Pro Gly Pro             420                 425                 430         Ile Asn Asp Asp Asp Asn Pro Gly His Gln Asp Asp Asp Pro Thr Asp         435                 440                 445             Ser Gln Asp Thr Thr Ile Pro Asp Val Val Val Asp Pro Asp Asp Gly     450                 455                 460                 Ser Tyr Gly Glu Tyr Gln Ser Tyr Ser Glu Asn Gly Met Asn Ala Pro 465                 470                 475                 480 Asp Asp Leu Val Leu Phe Asp Leu Asp Glu Asp Asp Glu Asp Thr Lys                 485                 490                 495     Pro Val Pro Asn Arg Ser Thr Lys Gly Gly Gln Gln Lys Asn Ser Gln             500                 505                 510         Lys Gly Gln His Ile Glu Gly Arg Gln Thr Gln Ser Arg Pro Ile Gln         515                 520                 525             Asn Val Pro Gly Pro His Arg Thr Ile His His Ala Ser Ala Pro Leu     530                 535                 540                 Thr Asp Asn Asp Arg Arg Asn Glu Pro Ser Gly Ser Thr Ser Pro Arg 545                 550                 555                 560 Met Leu Thr Pro Ile Asn Glu Glu Ala Asp Pro Leu Asp Asp Ala Asp                 565                 570                 575     Asp Glu Thr Ser Ser Leu Pro Pro Leu Glu Ser Asp Asp Glu Glu Gln             580                 585                 590         Asp Arg Asp Gly Thr Ser Asn Arg Thr Pro Thr Val Ala Pro Pro Ala         595                 600                 605             Pro Val Tyr Arg Asp His Ser Glu Lys Lys Glu Leu Pro Gln Asp Glu     610                 615                 620                 Gln Gln Asp Gln Asp His Thr Gln Glu Ala Arg Asn Gln Asp Ser Asp 625                 630                 635                 640 Asn Thr Gln Ser Glu His Ser Phe Glu Glu Met Tyr Arg His Ile Leu                 645                 650                 655     Arg Ser Gln Gly Pro Phe Asp Ala Val Leu Tyr Tyr His Met Met Lys             660                 665                 670         Asp Glu Pro Val Val Phe Ser Thr Ser Asp Gly Lys Glu Tyr Thr Tyr         675                 680                 685             Pro Asp Ser Leu Glu Glu Glu Tyr Pro Pro Trp Leu Thr Glu Lys Glu     690                 695                 700                 Ala Met Asn Glu Glu Asn Arg Phe Val Thr Leu Asp Gly Gln Gln Phe 705                 710                 715                 720 Tyr Trp Pro Val Met Asn His Lys Asn Lys Phe Met Ala Ile Leu Gln                 725                 730                 735     His His Gln  <210> SEQ ID NO 6 <211> LENGTH: 676 <212> TYPE: PRT<213> ORGANISM: Zaire ebolavirus <400> SEQUENCE: 6Met Gly Val Thr Gly Ile Leu Gln Leu Pro Arg Asp Arg Phe Lys Arg 1               5                   10                  15      Thr Ser Phe Phe Leu Trp Val Ile Ile Leu Phe Gln Arg Thr Phe Ser             20                  25                  30          Ile Pro Leu Gly Val Ile His Asn Ser Thr Leu Gln Val Ser Asp Val         35                  40                  45              Asp Lys Leu Val Cys Arg Asp Lys Leu Ser Ser Thr Asn Gln Leu Arg     50                  55                  60                  Ser Val Gly Leu Asn Leu Glu Gly Asn Gly Val Ala Thr Asp Val Pro 65                  70                  75                  80  Ser Ala Thr Lys Arg Trp Gly Phe Arg Ser Gly Val Pro Pro Lys Val                 85                  90                  95      Val Asn Tyr Glu Ala Gly Glu Trp Ala Glu Asn Cys Tyr Asn Leu Glu             100                 105                 110         Ile Lys Lys Pro Asp Gly Ser Glu Cys Leu Pro Ala Ala Pro Asp Gly         115                 120                 125             Ile Arg Gly Phe Pro Arg Cys Arg Tyr Val His Lys Val Ser Gly Thr     130                 135                 140                 Gly Pro Cys Ala Gly Asp Phe Ala Phe His Lys Glu Gly Ala Phe Phe 145                 150                 155                 160 Leu Tyr Asp Arg Leu Ala Ser Thr Val Ile Tyr Arg Gly Thr Thr Phe                 165                 170                 175     Ala Glu Gly Val Val Ala Phe Leu Ile Leu Pro Gln Ala Lys Lys Asp             180                 185                 190         Phe Phe Ser Ser His Pro Leu Arg Glu Pro Val Asn Ala Thr Glu Asp         195                 200                 205             Pro Ser Ser Gly Tyr Tyr Ser Thr Thr Ile Arg Tyr Gln Ala Thr Gly     210                 215                 220                 Phe Gly Thr Asn Glu Thr Glu Tyr Leu Phe Glu Val Asp Asn Leu Thr 225                 230                 235                 240 Tyr Val Gln Leu Glu Ser Arg Phe Thr Pro Gln Phe Leu Leu Gln Leu                 245                 250                 255     Asn Glu Thr Ile Tyr Thr Ser Gly Lys Arg Ser Asn Thr Thr Gly Lys             260                 265                 270         Leu Ile Trp Lys Val Asn Pro Glu Ile Asp Thr Thr Ile Gly Glu Trp         275                 280                 285             Ala Phe Trp Glu Thr Lys Lys Asn Leu Thr Arg Lys Ile Arg Ser Glu     290                 295                 300                 Glu Leu Ser Phe Thr Val Val Ser Asn Gly Ala Lys Asn Ile Ser Gly 305                 310                 315                 320 Gln Ser Pro Ala Arg Thr Ser Ser Asp Pro Gly Thr Asn Thr Thr Thr                 325                 330                 335     Glu Asp His Lys Ile Met Ala Ser Glu Asn Ser Ser Ala Met Val Gln             340                 345                 350         Val His Ser Gln Gly Arg Glu Ala Ala Val Ser His Leu Thr Thr Leu         355                 360                 365             Ala Thr Ile Ser Thr Ser Pro Gln Ser Leu Thr Thr Lys Pro Gly Pro     370                 375                 380                 Asp Asn Ser Thr His Asn Thr Pro Val Tyr Lys Leu Asp Ile Ser Glu 385                 390                 395                 400 Ala Thr Gln Val Glu Gln His His Arg Arg Thr Asp Asn Asp Ser Thr                 405                 410                 415     Ala Ser Asp Thr Pro Ser Ala Thr Thr Ala Ala Gly Pro Pro Lys Ala             420                 425                 430         Glu Asn Thr Asn Thr Ser Lys Ser Thr Asp Phe Leu Asp Pro Ala Thr         435                 440                 445             Thr Thr Ser Pro Gln Asn His Ser Glu Thr Ala Gly Asn Asn Asn Thr     450                 455                 460                 His His Gln Asp Thr Gly Glu Glu Ser Ala Ser Ser Gly Lys Leu Gly 465                 470                 475                 480 Leu Ile Thr Asn Thr Ile Ala Gly Val Ala Gly Leu Ile Thr Gly Gly                 485                 490                 495     Arg Arg Thr Arg Arg Glu Ala Ile Val Asn Ala Gln Pro Lys Cys Asn             500                 505                 510         Pro Asn Leu His Tyr Trp Thr Thr Gln Asp Glu Gly Ala Ala Ile Gly         515                 520                 525             Leu Ala Trp Ile Pro Tyr Phe Gly Pro Ala Ala Glu Gly Ile Tyr Ile     530                 535                 540                 Glu Gly Leu Met His Asn Gln Asp Gly Leu Ile Cys Gly Leu Arg Gln 545                 550                 555                 560 Leu Ala Asn Glu Thr Thr Gln Ala Leu Gln Leu Phe Leu Arg Ala Thr                 565                 570                 575     Thr Glu Leu Arg Thr Phe Ser Ile Leu Asn Arg Lys Ala Ile Asp Phe             580                 585                 590         Leu Leu Gln Arg Trp Gly Gly Thr Cys His Ile Leu Gly Pro Asp Cys         595                 600                 605             Cys Ile Glu Pro His Asp Trp Thr Lys Asn Ile Thr Asp Lys Ile Asp     610                 615                 620                 Gln Ile Ile His Asp Phe Val Asp Lys Thr Leu Pro Asp Gln Gly Asp 625                 630                 635                 640 Asn Asp Asn Trp Trp Thr Gly Trp Arg Gln Trp Ile Pro Ala Gly Ile                 645                 650                 655     Gly Val Thr Gly Val Ile Ile Ala Val Ile Ala Leu Phe Cys Ile Cys             660                 665                 670         Lys Phe Val Phe          675     

1. (canceled)
 2. A method for detecting the presence of a virus in ablood sample obtained from a subject, or of detecting prior contact bythe subject with said virus, the virus having viral nucleic acid,comprising: i) Performing a nucleic acid assay for detecting thepresence of, or measuring the amount of, the virus in said blood sampleobtained from a subject suspected of suffering from the viral disease,the assay comprising contacting a primer complementary to at least aportion of said viral nucleic acid with said sample; ii) performing anassay for detecting the presence of, or measuring the amount of,antibodies to the virus, in a sample obtained from a subject suspectedof suffering from the viral disease, the assay comprising contacting aviral antigen with said sample; iii) performing electrolyte assays formeasuring the amount of sodium and for measuring the amount of potassiumin the blood sample; and iv) providing or managing the treatment of thesubject guided by said measured amounts of sodium and potassium; whereinthe presence of the virus is detected in the occurrence of detection ofviral nucleic acid, or prior contact by the subject with the virus isdetected if antibodies to the virus are detected in the sample, andmanagement of the treatment of a subject suffering from an infectiousdisease is aided by c) measurement of abnormally low levels of sodium ord) measurement of abnormally high levels of potassium, in the bloodsample.
 3. The method of claim 2, wherein said blood sample is a smallvolume blood sample having a volume of less than about 250 microliters(μL).
 4. The method of claim 2, wherein said blood sample is afingerstick blood sample.
 5. The method of claim 2, wherein said nucleicacid assay comprises an isothermal nucleic acid assay.
 6. The method ofclaim 2, wherein said assay for detecting the presence of, or measuringthe amount of, antibodies to the virus, comprises IgM and IgG assays forantibodies to the virus. 7-8. (canceled)
 9. The method of claim 2,wherein said nucleic acid assay provides results within a short time ofless than about 1 hour and said assay for detecting the presence of, ormeasuring the amount of, antibodies to the virus provides results withina short time of less than about 1 hour.
 10. The method of claim 2,wherein said blood sample comprises a sample of EDTA-anti-coagulatedwhole blood.
 11. The method of claim 2, wherein said blood samplecomprises a sample of heparin-anti-coagulated plasma. 12-18. (canceled)19. An automatic sample analysis device configured to perform thefollowing method at a point of service location: i) Performing a nucleicacid assay for detecting the presence of, or measuring the amount of,the virus in said blood sample obtained from a subject suspected ofsuffering from the viral disease, the assay comprising contacting aprimer complementary to at least a portion of said viral nucleic acidwith said sample; ii) performing an assay for detecting the presence of,or measuring the amount of, antibodies to the virus, in a sampleobtained from a subject suspected of suffering from the viral disease,the assay comprising contacting a viral antigen with said sample; iii)performing electrolyte assays for measuring the amount of sodium and formeasuring the amount of potassium in the blood sample; and iv) providingor managing the treatment of the subject guided by said measured amountsof sodium and potassium; wherein the presence of the virus is detectedin the occurrence of detection of viral nucleic acid, or prior contactby the subject with the virus is detected if antibodies to the virus aredetected in the sample, and management of the treatment of a subjectsuffering from an infectious disease is aided by c) measurement ofabnormally low levels of sodium or d) measurement of abnormally highlevels of potassium, in the blood sample.
 20. The automatic sampleanalysis system of claim 19, further comprising a cartridge containingreagents for performing the methods of claim
 19. 21-25. (canceled)
 26. Acartridge comprising: a first polynucleotide primer and a secondpolynucleotide primer, wherein the first primer and the second primerare complementary to a nucleotide sequence from the Ebola virus, or to acomplementary sequence thereof; an anti-human IgM antibody; ananti-human IgG antibody; an isolated Ebola polypeptide antigen; and anisolated DNA polymerase having strand displacement activity.
 27. Thecartridge of claim 26, wherein the DNA polymerase is BstI.
 28. Thecartridge of claim 26, wherein at least one of the anti-human IgMantibody of the anti-human IgG antibody is immobilized on a solidsurface.
 29. (canceled)
 30. The cartridge of claim 26, wherein thecartridge further comprises reagents for the detection of one or both ofsodium or potassium ions.
 31. The cartridge of claim 30, wherein thecartridge comprises β-galactosidase for the detection of sodium ions.32. The cartridge of claim 30, wherein the cartridge comprises sodiumtetraphenylborate for the detection of potassium ions.
 33. The cartridgeof claim 30, wherein the cartridge comprises β-galactosidase and sodiumtetraphenylborate.
 34. The cartridge of claim 26, wherein the cartridgefurther comprises reagents for the detection of one or both of iron orhemoglobin.
 35. The cartridge of claim 26, wherein the cartridge furthercomprises a reagent to render a virus non-infectious.
 36. (canceled) 37.The cartridge of claim 26, wherein the cartridge further comprises ananti-Ebola virus antigen antibody.
 38. (canceled)